Enzyme-Expressing Yeast For Ethanol Production

ABSTRACT

Described herein are recombinant fermenting organisms having a heterologous polynucleotide encoding a phospholipase. Also described are processes for producing a fermentation product, such as ethanol, from starch or cellulosic-containing material with the recombinant fermenting organisms.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND

Production of ethanol from starch and cellulosic containing materials iswell-known in the art.

The most commonly industrially used commercial process forstarch-containing material, often referred to as a “conventionalprocess”, includes liquefying gelatinized starch at high temperature(about 85° C.) using typically a bacterial alpha-amylase, followed bysimultaneous saccharification and fermentation (SSF) carried outanaerobically in the presence of typically a glucoamylase and aSaccharomyces cerevisae yeast.

Yeasts which are used for production of ethanol for use as fuel, such asin the corn ethanol industry, require several characteristics to ensurecost effective production of the ethanol. These characteristics includeethanol tolerance, low by-product yield, rapid fermentation, and theability to limit the amount of residual sugars remaining in the ferment.Such characteristics have a marked effect on the viability of theindustrial process.

Yeast of the genus Saccharomyces exhibits many of the characteristicsrequired for production of ethanol. In particular, strains ofSaccharomyces cerevisiae are widely used for the production of ethanolin the fuel ethanol industry. Strains of Saccharomyces cerevisiae thatare widely used in the fuel ethanol industry have the ability to producehigh yields of ethanol under fermentation conditions found in, forexample, the fermentation of corn mash. An example of such a strain isthe yeast used in commercially available ethanol yeast product calledETHANOL RED®.

Saccharomyces cerevisae yeast have been genetically engineered toexpress alpha-amylase and/or glucoamylase to improve yield and decreasethe amount of exogenously added enzymes necessary during SSF (e.g.,WO2018/098381, WO2017/087330, WO2017/037614, WO2011/128712,WO2011/153516, US2018/0155744). Yeast have also been engineered toexpress trehalase in an attempt to increase fermentation yield bybreaking down residual trehalose (e.g., WO2017/077504).

WO2008/135547 concerns reducing foam in processes for production of afermentation product by contacting the fermentation media comprising afermenting organism with a lipolytic enzyme selected from the groupconsisting of phospholipase, lyso-phospholipase and lipase, and a metalsalt.

WO2014/147219 concerns a phospholipase A from Talaromyces leycettanus.

WO2015/140275 discloses a phospholipase C from Bacillus thuringiensis.

Despite significant improvement of ethanol production processes over thepast decade there is still a desire and need for providing improvedprocesses of ethanol fermentation from starch and cellulosic containingmaterial in an economically and commercially relevant scale.

For example, foam generation during ethanol fermentation is a majorproblem, especially in ethanol production processes wherestarch-containing material is liquefied with an alpha-amylase and aprotease before saccharification and fermentation. Additionally, the useof nitrogen supplements (e.g., urea) is an added expense duringfermentation. Therefore, there is a desire to, inter alia, reduce foamand/or reduce supplemental nitrogen requirements in ethanolfermentation.

SUMMARY

Described herein are, inter alia, methods for producing a fermentationproduct, such as ethanol, from starch or cellulosic-containing material,and yeast suitable for use in such processes. The Applicant hassurprisingly found that yeasts expressing a phospholipase providebeneficial properties during fermentation, such as reduced foaming,improved oil extraction yield, and improved ethanol yield.

A first aspect relates to methods of producing a fermentation productfrom a starch-containing or cellulosic-containing material comprising:(a) saccharifying the starch-containing or cellulosic-containingmaterial; and (b) fermenting the saccharified material of step (a) witha fermenting organism; wherein the fermenting organism comprises aheterologous polynucleotide encoding a phospholipase. In someembodiments, the phospholipase is a Phospholipase A or a PhospholipaseC.

In some embodiments of the methods, fermentation and saccharificationare performed simultaneously in a simultaneous saccharification andfermentation (SSF). In other embodiments, fermentation andsaccharification are performed sequentially (SHF).

In some embodiments of the methods, the method comprises recovering thefermentation product from the from the fermentation (e.g., bydistillation).

In some embodiments of the methods, the fermentation product is ethanol.

In some embodiments of the methods, fermentation is performed underreduced nitrogen conditions (e.g., less than 1000 ppm urea or ammoniumhydroxide, such as less than 750 ppm, less than 500 ppm, less than 400ppm, less than 300 ppm, less than 250 ppm, less than 200 ppm, less than150 ppm, less than 100 ppm, less than 75 ppm, less than 50 ppm, lessthan 25 ppm, or less than 10 ppm).

In some embodiments of the methods, the method results in higher yieldof fermentation product (e.g., ethanol) and/or reduced foam accumulationwhen compared to the same process using an identical cell without theheterologous polynucleotide encoding the phospholipase under the sameconditions (e.g., at about or after 54 hours fermentation, such as theconditions described in Examples 3 or 4). In some embodiments, themethod results in at least 0.25% (e.g., 0.5%, 0.75%, 1.0%, 1.25%, 1.5%,1.75%, 2%, 3% or 5%) higher yield of fermentation product.

In some embodiments of the methods, the phospholipase has a maturepolypeptide sequence with 60%, e.g., at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100% sequence identity, to the amino acidsequence of any one of SEQ ID NOs: 235-242 and 252-342. In someembodiments of the methods, the phospholipase has a mature polypeptidesequence with 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence ofany one of SEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and 242. Insome embodiments of the methods, the heterologous polynucleotide encodesa phospholipase having a mature polypeptide sequence that differs by nomore than ten amino acids, e.g., by no more than five amino acids, by nomore than four amino acids, by no more than three amino acids, by nomore than two amino acids, or by one amino acid from the amino acidsequence of any one of SEQ ID NOs: 235-242 and 252-342 (e.g., any one ofSEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and 242). In someembodiments of the methods, the heterologous polynucleotide encodes aphospholipase having a mature polypeptide sequence comprising orconsisting of the amino acid sequence of any one of SEQ ID NOs: 235-242and 252-342 (e.g., any one of SEQ ID NOs: SEQ ID NOs: 235, 236, 237,238, 239, 240, 241 and 242).

In some embodiments of the methods, saccharification of step occurs on astarch-containing material, and wherein the starch-containing materialis either gelatinized or ungelatinized starch.

In some embodiments of the methods, the method comprises liquefying thestarch-containing material by contacting the material with analpha-amylase prior to saccharification.

In some embodiments of the methods, liquefying the starch-containingmaterial and/or saccharifying the starch-containing material isconducted in presence of exogenously added protease.

In some embodiments of the methods, the fermenting organism comprises aheterologous polynucleotide encoding a glucoamylase, such as aglucoamylase having a mature polypeptide sequence with 60%, e.g., atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity, to the amino acid sequence of a Pycnoporus glycoamylase (e.g.,a Pycnoporus sanguineus glucoamylase of SEQ ID NO: 229), a Gloeophyllumglucoamylase (e.g. a Gloeophyllum sepiarium of SEQ ID NO: 8), or aglucoamylase of any one of SEQ ID NOs: 102-113 (e.g., a Saccharomycopsisfibuligera glucoamylase of SEQ ID NO: 103 or 104, or a Trichodermareesei glucoamylase of SEQ ID NO: 230).

In some embodiments of the methods, the fermenting organism comprises aheterologous polynucleotide encoding an alpha-amylase, such as analpha-amylase having a mature polypeptide sequence with 60%, e.g., atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity, to the amino acid sequence of any one of SEQ ID NOs: 76-101,121-174 and 231. In some embodiments of the methods, the heterologouspolynucleotide encodes an alpha-amylase having a mature polypeptidesequence that differs by no more than ten amino acids, e.g., by no morethan five amino acids, by no more than four amino acids, by no more thanthree amino acids, by no more than two amino acids, or by one amino acidfrom the amino acid sequence of any one of SEQ ID NOs: 76-101, 121-174and 231. In some embodiments of the methods, the heterologouspolynucleotide encodes an alpha-amylase having a mature polypeptidesequence comprising or consisting of the amino acid sequence of any oneof SEQ ID NOs: SEQ ID NOs: 76-101, 121-174 and 231.

In some embodiments of the methods, the fermenting organism comprises aheterologous polynucleotide encoding a trehalase, such as a trehalasehaving a mature polypeptide sequence with 60%, e.g., at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity, tothe amino acid sequence of any one of SEQ ID NOs: 175-226. In someembodiments of the methods, the heterologous polynucleotide encodes atrehalase having a mature polypeptide sequence that differs by no morethan ten amino acids, e.g., by no more than five amino acids, by no morethan four amino acids, by no more than three amino acids, by no morethan two amino acids, or by one amino acid from the amino acid sequenceof any one of SEQ ID NOs: 175-226. In some embodiments of the methods,the heterologous polynucleotide encodes a trehalase having a maturepolypeptide sequence comprising or consisting of the amino acid sequenceof any one of SEQ ID NOs: SEQ ID NOs: 175-226.

In some embodiments of the methods, the fermenting organism comprises aheterologous polynucleotide encoding a protease, such as a proteasehaving a mature polypeptide sequence of at least 60%, e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs: 9-73(e.g., any one of SEQ ID NOs: 9, 14, 16, 21, 22, 33, 41, 45, 61, 62, 66,67, and 69; such as any one of SEQ NOs: 9, 14, 16, and 69).

In some embodiments of the methods, saccharification of step occurs on acellulosic-containing material, and wherein the cellulosic-containingmaterial is pretreated (e.g. a dilute acid pretreatment).

In some embodiments of the methods, saccharification occurs on acellulosic-containing material, and wherein the enzyme compositioncomprises one or more enzymes selected from a cellulase (e.g.,endoglucanase, a cellobiohydrolase, or a beta-glucosidase), an AA9polypeptide, a hemicellulase (e.g., a xylanase, an acetylxylan esterase,a feruloyl esterase, an arabinofuranosidase, a xylosidase, or aglucuronidase), a CIP, an esterase, an expansin, a ligninolytic enzyme,an oxidoreductase, a pectinase, a protease, and a swollenin.

In some embodiments of the methods, the fermenting organism is aSaccharomyces, Rhodotorula, Schizosaccharomyces, Kluyveromyces, Pichia,Hansenula, Rhodosporidium, Candida, Yarrowia, Lipomyces, Cryptococcus,or Dekkera sp. cell. In some embodiments, the fermenting organism is aSaccharomyces cerevisiae cell.

Another aspect relates to a recombinant yeast cell comprising aheterologous polynucleotide encoding a phospholipase. In someembodiments, the phospholipase is a Phospholipase A or a PhospholipaseC.

In some embodiments of the yeast cell, the cell is capable of higheryield of fermentation product and/or reduced foam accumulation whencompared to fermentation using the same process and an identical cellwithout the heterologous polynucleotide encoding the phospholipase underthe same conditions (e.g., at about or after 54 hours fermentation, suchas the conditions described in Examples 3 or 4). In some embodiments,the cell is capable of at least 0.25% (e.g., 0.5%, 0.75%, 1.0%, 1.25%,1.5%, 1.75%, 2%, 3% or 5%) higher yield of fermentation product.

In some embodiments, the recombinant yeast cell is a Saccharomyces,Rhodotorula, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula,Rhodosporidium, Candida, Yarrowia, Lipomyces, Cryptococcus, or Dekkerasp. cell. In some embodiments, the recombinant yeast cell is aSaccharomyces cerevisiae cell.

In some embodiments of the yeast cell, the fermenting organism comprisesa heterologous polynucleotide encoding a glucoamylase, such as aglucoamylase having a mature polypeptide sequence with 60%, e.g., atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity, to the amino acid sequence of a Pycnoporus glycoamylase (e.g.,a Pycnoporus sanguineus glucoamylase of SEQ ID NO: 229), a Gloeophyllumglucoamylase (e.g. a Gloeophyllum sepiarium of SEQ ID NO: 8), or aglucoamylase of any one of SEQ ID NOs: 102-113 (e.g., a Saccharomycopsisfibuligera glucoamylase of SEQ ID NO: 103 or 104, or a Trichodermareesei glucoamylase of SEQ ID NO: 230).

In some embodiments of the yeast cell, the fermenting organism comprisesa heterologous polynucleotide encoding an alpha-amylase, wherein thealpha-amylase has a mature polypeptide sequence with 60%, e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity, to the amino acid sequence of any one of SEQ ID NOs: 76-101,121-174 and 231. In some embodiments of the methods, the heterologouspolynucleotide encodes an alpha-amylase having a mature polypeptidesequence that differs by no more than ten amino acids, e.g., by no morethan five amino acids, by no more than four amino acids, by no more thanthree amino acids, by no more than two amino acids, or by one amino acidfrom the amino acid sequence of any one of SEQ ID NOs: 76-101, 121-174and 231. In some embodiments of the methods, the heterologouspolynucleotide encodes an alpha-amylase having a mature polypeptidesequence comprising or consisting of the amino acid sequence of any oneof SEQ ID NOs: SEQ ID NOs: 76-101, 121-174 and 231.

In some embodiments of the yeast cell, the fermenting organism comprisesa heterologous polynucleotide encoding a trehalase, wherein thetrehalase has mature polypeptide sequence with 60%, e.g., at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity,to the amino acid sequence of any one of SEQ ID NOs: 175-226. In someembodiments of the methods, the heterologous polynucleotide encodes atrehalase having a mature polypeptide sequence that differs by no morethan ten amino acids, e.g., by no more than five amino acids, by no morethan four amino acids, by no more than three amino acids, by no morethan two amino acids, or by one amino acid from the amino acid sequenceof any one of SEQ ID NOs: 175-226. In some embodiments of the methods,the heterologous polynucleotide encodes a trehalase having a maturepolypeptide sequence comprising or consisting of the amino acid sequenceof any one of SEQ ID NOs: SEQ ID NOs: 175-226.

In some embodiments of the yeast cell, the fermenting organism comprisesa heterologous polynucleotide encoding a protease, such as a proteasehaving a mature polypeptide sequence of at least 60%, e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs: 9-73(e.g., any one of SEQ ID NOs: 9, 14, 16, 21, 22, 33, 41, 45, 61, 62, 66,67, and 69; such as any one of SEQ NOs: 9, 14, 16, and 69).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows % ethanol improvement for phoshoplipase-expressing yeaststrains and control strain MHCT-484 described in Example 3.

FIG. 2 shows normalized mean ethanol improvement after 54 hours offermentation of AMP mash at 0 and 150 ppm urea as described in Example4.

FIG. 3 shows normalized mean ethanol improvement after 54 hours offermentation of the non-AMP mash at 0 and 300 ppm urea as described inExample 4.

FIG. 4 shows improved defoaming capability during fermentation of aphospholipase-expressing yeast strain HP21-F04 (right) compared tocontrol strain yMHCT48 (left) as described in Example 9.

DEFINITIONS

Unless defined otherwise or clearly indicated by context, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-amylase: The term “alpha amylase” means an 1,4-alpha-D-glucanglucanohydrolase, EC. 3.2.1.1, which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides. Forpurposes of the present invention, alpha amylase activity can bedetermined using an alpha amylase assay described in the examplessection below.

Auxiliary Activity 9: The term “Auxiliary Activity 9” or “AA9” means apolypeptide classified as a lytic polysaccharide monooxygenase (Quinlanet al., 2011, Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips etal., 2011, ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20:1051-1061). AA9 polypeptides were formerly classified into the glycosidehydrolase Family 61 (GH61) according to Henrissat, 1991, Biochem. J.280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

AA9 polypeptides enhance the hydrolysis of a cellulosic-containingmaterial by an enzyme having cellulolytic activity. Cellulolyticenhancing activity can be determined by measuring the increase inreducing sugars or the increase of the total of cellobiose and glucosefrom the hydrolysis of a cellulosic-containing material by cellulolyticenzyme under the following conditions: 1-50 mg of total protein/g ofcellulose in pretreated corn stover (PCS), wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of an AA9 polypeptide for 1-7 days at a suitable temperature,such as 40 C-80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C.,and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, or 8.5, compared to a control hydrolysis with equal total proteinloading without cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS).

AA9 polypeptide enhancing activity can be determined using a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsvrd, Denmark) and beta-glucosidaseas the source of the cellulolytic activity, wherein the beta-glucosidaseis present at a weight of at least 2-5% protein of the cellulase proteinloading. In one embodiment, the beta-glucosidase is an Aspergillusoryzae beta-glucosidase (e.g., recombinantly produced in Aspergillusoryzae according to WO 02/095014). In another embodiment, thebeta-glucosidase is an Aspergillus fumigatus beta-glucosidase (e.g.,recombinantly produced in Aspergillus oryzae as described in WO02/095014).

AA9 polypeptide enhancing activity can also be determined by incubatingan AA9 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC),100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/mL ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hoursat 40° C. followed by determination of the glucose released from thePASC.

AA9 polypeptide enhancing activity can also be determined according toWO 2013/028928 for high temperature compositions.

AA9 polypeptides enhance the hydrolysis of a cellulosic-containingmaterial catalyzed by enzyme having cellulolytic activity by reducingthe amount of cellulolytic enzyme required to reach the same degree ofhydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, atleast 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, orat least 20-fold.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity can be determined usingp-nitrophenyl-beta-D-glucopyranoside as substrate according to theprocedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. Oneunit of beta-glucosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. Beta-xylosidase activity can be determinedusing 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20 at pH 5, 40° C. One unit ofbeta-xylosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01%TWEEN® 20.

Catalase: The term “catalase” means ahydrogen-peroxide:hydrogen-peroxide oxidoreductase (EC 1.11.1.6) thatcatalyzes the conversion of 2 H₂O₂ to O₂+2 H₂O. For purposes of thepresent invention, catalase activity is determined according to U.S.Pat. No. 5,646,025. One unit of catalase activity equals the amount ofenzyme that catalyzes the oxidation of 1 μmole of hydrogen peroxideunder the assay conditions.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity canbe determined according to the procedures described by Lever et al.,1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic-containing material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanN21 filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No.1 filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increasein production/release of sugars during hydrolysis of acellulosic-containing material by cellulolytic enzyme(s) under thefollowing conditions: 1-50 mg of cellulolytic enzyme protein/g ofcellulose in pretreated corn stover (PCS) (or other pretreatedcellulosic-containing material) for 3-7 days at a suitable temperaturesuch as 40° C.-80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C.,and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0,compared to a control hydrolysis without addition of cellulolytic enzymeprotein. Typical conditions are 1 mL reactions, washed or unwashed PCS,5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnSO₄,50° C., 55° C., or 60° C., 72 hours, sugar analysis by AMINEX® HPX-87Hcolumn chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA).

Coding sequence: The term “coding sequence” or “coding region” means apolynucleotide sequence, which specifies the amino acid sequence of apolypeptide. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea sequence of genomic DNA, cDNA, a synthetic polynucleotide, and/or arecombinant polynucleotide.

Control sequence: The term “control sequence” means a nucleic acidsequence necessary for polypeptide expression. Control sequences may benative or foreign to the polynucleotide encoding the polypeptide, andnative or foreign to each other. Such control sequences include, but arenot limited to, a leader sequence, polyadenylation sequence, propeptidesequence, promoter sequence, signal peptide sequence, and transcriptionterminator sequence. The control sequences may be provided with linkersfor the purpose of introducing specific restriction sites facilitatingligation of the control sequences with the coding region of thepolynucleotide encoding a polypeptide.

Disruption: The term “disruption” means that a coding region and/orcontrol sequence of a referenced gene is partially or entirely modified(such as by deletion, insertion, and/or substitution of one or morenucleotides) resulting in the absence (inactivation) or decrease inexpression, and/or the absence or decrease of enzyme activity of theencoded polypeptide. The effects of disruption can be measured usingtechniques known in the art such as detecting the absence or decrease ofenzyme activity using from cell-free extract measurements referencedherein; or by the absence or decrease of corresponding mRNA (e.g., atleast 25% decrease, at least 50% decrease, at least 60% decrease, atleast 70% decrease, at least 80% decrease, or at least 90% decrease);the absence or decrease in the amount of corresponding polypeptidehaving enzyme activity (e.g., at least 25% decrease, at least 50%decrease, at least 60% decrease, at least 70% decrease, at least 80%decrease, or at least 90% decrease); or the absence or decrease of thespecific activity of the corresponding polypeptide having enzymeactivity (e.g., at least 25% decrease, at least 50% decrease, at least60% decrease, at least 70% decrease, at least 80% decrease, or at least90% decrease). Disruptions of a particular gene of interest can begenerated by methods known in the art, e.g., by directed homologousrecombination (see Methods in Yeast Genetics (1997 edition), Adams,Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998)).

Endogenous gene: The term “endogenous gene” means a gene that is nativeto the referenced host cell. “Endogenous gene expression” meansexpression of an endogenous gene.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). Endoglucanase activity can also bedetermined using carboxymethyl cellulose (CMC) as substrate according tothe procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5,40° C.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion. Expression can bemeasured—for example, to detect increased expression—by techniques knownin the art, such as measuring levels of mRNA and/or translatedpolypeptide.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fermentable medium: The term “fermentable medium” or “fermentationmedium” refers to a medium comprising one or more (e.g., two, several)sugars, such as glucose, fructose, sucrose, cellobiose, xylose,xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides, wherein the medium is capable, in part, of beingconverted (fermented) by a host cell into a desired product, such asethanol. In some instances, the fermentation medium is derived from anatural source, such as sugar cane, starch, or cellulose, and may be theresult of pretreating the source by enzymatic hydrolysis(saccharification). The term fermentation medium is understood herein torefer to a medium before the fermenting organism is added, such as, amedium resulting from a saccharification process, as well as a mediumused in a simultaneous saccharification and fermentation process (SSF).

Glucoamylase: The term “glucoamylase” (1,4-alpha-D-glucanglucohydrolase, EC 3.2.1.3) is defined as an enzyme that catalyzes therelease of D-glucose from the non-reducing ends of starch or relatedoligo- and polysaccharide molecules. For purposes of the presentinvention, glucoamylase activity may be determined according to theprocedures known in the art, such as those described in the Examples ofU.S. Provisional Patent Application No. 62/703,103, filed Jul. 25, 2018.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Current Opinion In Microbiology 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40° C.-80° C., e.g., 50°C., 55° C., 60° C., 65° C., or 70° C., and a suitable pH such as 4-9,e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.

Heterologous polynucleotide: The term “heterologous polynucleotide” isdefined herein as a polynucleotide that is not native to the host cell;a native polynucleotide in which structural modifications have been madeto the coding region; a native polynucleotide whose expression isquantitatively altered as a result of a manipulation of the DNA byrecombinant DNA techniques, e.g., a different (foreign) promoter; or anative polynucleotide in a host cell having one or more extra copies ofthe polynucleotide to quantitatively alter expression. A “heterologousgene” is a gene comprising a heterologous polynucleotide.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotidedescribed herein (e.g., a polynucleotide encoding an alpha-amylaseand/or trehalase). The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The term “recombinant cell” is definedherein as a non-naturally occurring host cell comprising one or more(e.g., two, several) heterologous polynucleotides.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 50° C.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having biological activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. The mature polypeptide sequence lacks a signalsequence, which may be determined using techniques known in the art(See, e.g., Zhang and Henzel, 2004, Protein Science 13: 2819-2824).

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mLsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/mL sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means apolynucleotide comprises one or more (e.g., two, several) controlsequences. The polynucleotide may be single-stranded or double-stranded,and may be isolated from a naturally occurring gene, modified to containsegments of nucleic acids in a manner that would not otherwise exist innature, or synthetic.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Phospholipase: The term “phospholipase” means an enzyme that catalyzesthe conversion of phospholipids into fatty acids and other lipophilicsubstances, such as phospholipase A (EC numbers 3.1.1.4, 3.1.1.5 and3.1.1.32) or phospholipase C (EC numbers 3.1.4.3 and 3.1.4.11). Forpurposes of the present invention, phospholipase activity may bedetermined using activity assays known in the art, or according to theprocedures described in the Examples herein (Example 2).

Pretreated corn stover: The term “Pretreated Corn Stover” or “PCS” meansa cellulosic-containing material derived from corn stover by treatmentwith heat and dilute sulfuric acid, alkaline pretreatment, neutralpretreatment, or any pretreatment known in the art.

Protease: The term “protease” is defined herein as an enzyme thathydrolyses peptide bonds. It includes any enzyme belonging to the EC 3.4enzyme group (including each of the thirteen subclasses thereof). The ECnumber refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press,San Diego, Calif., including supplements 1-5 published in Eur. J.Biochem. 223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J.Biochem. 237: 1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J.Biochem. 264: 610-650 (1999); respectively. The term “subtilases” referto a sub-group of serine protease according to Siezen et al., 1991,Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6:501-523. Serine proteases or serine peptidases is a subgroup ofproteases characterised by having a serine in the active site, whichforms a covalent adduct with the substrate. Further the subtilases (andthe serine proteases) are characterised by having two active site aminoacid residues apart from the serine, namely a histidine and an asparticacid residue. The subtilases may be divided into 6 sub-divisions, i.e.the Subtilisin family, the Thermitase family, the Proteinase K family,the Lantibiotic peptidase family, the Kexin family and the Pyrolysinfamily. The term “protease activity” means a proteolytic activity (EC3.4). Protease activity may be determined using methods described in theart (e.g., US 2015/0125925) or using commercially available assay kits(e.g., Sigma-Aldrich).

Pullulanase: The term “pullulanase” means a starch debranching enzymehaving pullulan 6-glucano-hydrolase activity (EC 3.2.1.41) thatcatalyzes the hydrolysis the α-1,6-glycosidic bonds in pullulan,releasing maltotriose with reducing carbohydrate ends. For purposes ofthe present invention, pullulanase activity can be determined accordingto a PHADEBAS assay or the sweet potato starch assay described inWO2016/087237.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes described herein, the degree of sequence identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, J. Mol. Biol. 1970, 48, 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., TrendsGenet 2000, 16, 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of the Referenced Sequence−Total Numberof Gaps in Alignment)

For purposes described herein, the degree of sequence identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of ReferencedSequence−Total Number of Gaps in Alignment)

Signal peptide: The term “signal peptide” is defined herein as a peptidelinked (fused) in frame to the amino terminus of a polypeptide havingbiological activity and directs the polypeptide into the cell'ssecretory pathway. Signal sequences may be determined using techniquesknown in the art (See, e.g., Zhang and Henzel, 2004, Protein Science 13:2819-2824). The polypeptides described herein may comprise any suitablesignal peptide known in the art, or any signal peptide described herein(e.g., the S. cerevisiae MFα1 signal peptide of SEQ ID NO: 7, the S.cerevisiae EXG1 signal peptide of SEQ ID NO: 227, or the S. cerevisiaeAG2 signal peptide of SEQ ID NO: 234, or a signal peptide having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identitythereof).

Trehalase: The term “trehalase” means an enzyme which degrades trehaloseinto its unit monosaccharides (i.e., glucose). Trehalases are classifiedin EC 3.2.1.28 (alpha,alpha-trehalase) and EC. 3.2.1.93(alpha,alpha-phosphotrehalase). The EC classes are based onrecommendations of the Nomenclature Committee of the International Unionof Biochemistry and Molecular Biology (IUBMB). Description of EC classescan be found on the internet, e.g., on “http://www.expasy.org/enzyme/”.Trehalases are enzymes that catalyze the following reactions:

EC 3.2.1.28: Alpha,alpha-trehalose+H₂O

2 D-glucose;

EC 3.2.1.93: Alpha,alpha-trehalose 6-phosphate+H₂O

D-glucose+D-glucose 6-phosphate.

For purposes of the present invention, trehalase activity may bedetermined according to the trehalase activity assay described herein inthe experimental section.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/mL sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 45° C.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. Xylanase activity can be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Xylose Isomerase: The term “Xylose Isomerase” or “XI” means an enzymewhich can catalyze D-xylose into D-xylulose in vivo, and convertD-glucose into D-fructose in vitro. Xylose isomerase is also known as“glucose isomerase” and is classified as E.C. 5.3.1.5. As the structureof the enzyme is very stable, the xylose isomerase is a good model forstudying the relationships between protein structure and functions(Karimaki et al., Protein Eng Des Sel, 12004, 17 (12):861-869). XyloseIsomerase activity may be determined using techniques known in the art(e.g., a coupled enzyme assay using D-sorbitol dehygrogenase, asdescribed by Verhoeven et. al., 2017, Sci Rep 7, 46155).

Reference to “about” a value or parameter herein includes embodimentsthat are directed to that value or parameter per se. For example,description referring to “about X” includes the embodiment “X”. Whenused in combination with measured values, “about” includes a range thatencompasses at least the uncertainty associated with the method ofmeasuring the particular value, and can include a range of plus or minustwo standard deviations around the stated value.

Likewise, reference to a gene or polypeptide that is “derived from”another gene or polypeptide X, includes the gene or polypeptide X.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

It is understood that the embodiments described herein include“consisting” and/or “consisting essentially of” embodiments. As usedherein, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprise” or variationssuch as “comprises” or “comprising” is used in an inclusive sense, i.e.to specify the presence of the stated features but not to preclude thepresence or addition of further features in various embodiments.

DETAILED DESCRIPTION

Described herein, inter alia, are methods for producing a fermentationproduct, such as ethanol, from starch or cellulosic containing material.

During industrial scale fermentation, yeast encounter variousphysiological challenges including variable concentrations of sugars,high concentrations of yeast metabolites such as ethanol, glycerol,organic acids, osmotic stress, as well as potential competition fromcontaminating microbes such as wild yeasts and bacteria. As aconsequence, many yeasts are not suitable for use in industrialfermentation. The most widely used commercially available industrialstrain of Saccharomyces (i.e. for industrial scale fermentation) is theSaccharomyces cerevisiae strain used, for example, in the productETHANOL RED®. This strain is well suited to industrial ethanolproduction; however, it remains unclear how modifications to the yeastwill impact performance. In particular, the functional expression ofheterologous enzymes by an industrially-relevant Saccharomycescerevisiae yeast is uncertain (See, for example U.S. Pat. No. 9,206,444where the applicant was unable to functionally express numerousenzymes/enzyme classes).

The Applicant has surprisingly found that yeast expressing aphosholipase provide beneficial properties that may be useful forethanol fermentation, such as reduced need for supplemental nitrogen.

In one aspect is a method of producing a fermentation product from astarch-containing or cellulosic-containing material comprising:

(a) saccharifying the starch-containing or cellulosic-containingmaterial; and

(b) fermenting the saccharified material of step (a) with a fermentingorganism;

wherein the fermenting organism comprises a heterologous polynucleotideencoding a phospholipase.

Steps of saccharifying and fermenting are carried out eithersequentially or simultaneously (SSF). In one embodiment, steps ofsaccharifying and fermenting are carried out simultaneously (SSF). Inanother embodiment, steps of saccharifying and fermenting are carriedout sequentially.

Fermenting Organism

The fermenting organism described herein may be derived from any hostcell known to the skilled artisan capable of producing a fermentationproduct, such as ethanol. As used herein, a “derivative” of strain isderived from a referenced strain, such as through mutagenesis,recombinant DNA technology, mating, cell fusion, or cytoduction betweenyeast strains. Those skilled in the art will understand that the geneticalterations, including metabolic modifications exemplified herein, maybe described with reference to a suitable host organism and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art can apply the teachings and guidance provided hereinto other organisms. For example, the metabolic alterations exemplifiedherein can readily be applied to other species by incorporating the sameor analogous encoding nucleic acid from species other than thereferenced species.

The host cells for preparing the recombinant cells described herein canbe from any suitable host, such as a yeast strain, including, but notlimited to, a Saccharomyces, Rhodotorula, Schizosaccharomyces,Kluyveromyces, Pichia, Hansenula, Rhodosporidium, Candida, Yarrowia,Lipomyces, Cryptococcus, or Dekkera sp. cell. In particular,Saccharomyces host cells are contemplated, such as Saccharomycescerevisiae, bayanus or carlsbergensis cells. Preferably, the yeast cellis a Saccharomyces cerevisiae cell. Suitable cells can, for example, bederived from commercially available strains and polyploid or aneuploidindustrial strains, including but not limited to those from Superstart™,THERMOSACC®, C5 FUEL™, XyloFerm®, etc. (Lallemand); RED STAR and ETHANOLREDO (Fermentis/Lesaffre); FALI (AB Mauri); Baker's Best Yeast, Baker'sCompressed Yeast, etc. (Fleishmann's Yeast); BIOFERM AFT, XP, CF, and XR(North American Bioproducts Corp.); Turbo Yeast (Gert Strand AB); andFERMIOL® (DSM Specialties). Other useful yeast strains are availablefrom biological depositories such as the American Type CultureCollection (ATCC) or the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), such as, e.g., BY4741 (e.g., ATCC 201388);Y108-1 (ATCC PTA. 10567) and NRRL YB-1952 (ARS Culture Collection).Still other S. cerevisiae strains suitable as host cells DBY746,[Alpha][Eta]22, 5150-2B, GPY55-15Ba, CEN.PK, USM21, TMB3500, TMB3400,VTT-A-63015, VTT-A-85068, VTT-c-79093 and their derivatives as well asSaccharomyces sp. 1400, 424A (LNH-ST), 259A (LNH-ST) and derivativesthereof. In one embodiment, the recombinant cell is a derivative of astrain Saccharomyces cerevisiae CIBTS1260 (deposited under Accession No.NRRL Y-50973 at the Agricultural Research Service Culture Collection(NRRL), Illinois 61604 U.S.A.).

The fermenting organism may be Saccharomyces strain, e.g., Saccharomycescerevisiae strain produced using the method described and concerned inU.S. Pat. No. 8,257,959-BB.

The strain may also be a derivative of Saccharomyces cerevisiae strainNMI V14/004037 (See, WO2015/143324 and WO2015/143317 each incorporatedherein by reference), strain nos. V15/004035, V15/004036, and V15/004037(See, WO 2016/153924 incorporated herein by reference), strain nos.V15/001459, V15/001460, V15/001461 (See, WO2016/138437 incorporatedherein by reference), strain no. NRRL Y67342 (See, WO2017/063159incorporated herein by reference), or any strain described inWO2017/087330 (incorporated herein by reference).

The fermenting organisms according to the invention have been generatedin order to, e.g., improve fermentation yield and to improve processeconomy by cutting enzyme costs since part or all of the necessaryenzymes needed to improve method performance are be produced by thefermenting organism.

The fermenting organisms described herein may utilize expression vectorscomprising the coding sequence of one or more (e.g., two, several)heterologous genes linked to one or more control sequences that directexpression in a suitable cell under conditions compatible with thecontrol sequence(s). Such expression vectors may be used in any of thecells and methods described herein. The polynucleotides described hereinmay be manipulated in a variety of ways to provide for expression of adesired polypeptide. Manipulation of the polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

A construct or vector (or multiple constructs or vectors) comprising theone or more (e.g., two, several) heterologous genes may be introducedinto a cell so that the construct or vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier.

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or more(e.g., two, several) convenient restriction sites to allow for insertionor substitution of the polynucleotide at such sites.

Alternatively, the polynucleotide(s) may be expressed by inserting thepolynucleotide(s) or a nucleic acid construct comprising the sequenceinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the cell, or atransposon, may be used.

The expression vector may contain any suitable promoter sequence that isrecognized by a cell for expression of a gene described herein. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anypolynucleotide that shows transcriptional activity in the cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the cell.

Each heterologous polynucleotide described herein may be operably linkedto a promoter that is foreign to the polynucleotide. For example, in oneembodiment, the heterologous polynucleotide encoding the hexosetransporter is operably linked to a promoter foreign to thepolynucleotide. The promoters may be identical to or share a high degreeof sequence identity (e.g., at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) with a selected nativepromoter.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in a yeast cells, include, but are not limitedto, the promoters obtained from the genes for enolase, (e.g., S.cerevisiae enolase or I. orientalis enolase (ENO1)), galactokinase(e.g., S. cerevisiae galactokinase or I. orientalis galactokinase(GAL1)), alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(e.g., S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase or I. orientalis alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP)), triose phosphate isomerase (e.g., S. cerevisiae triosephosphate isomerase or I. orientalis triose phosphate isomerase (TPI)),metallothionein (e.g., S. cerevisiae metallothionein or I. orientalismetallothionein (CUP1)), 3-phosphoglycerate kinase (e.g., S. cerevisiae3-phosphoglycerate kinase or I. orientalis 3-phosphoglycerate kinase(PGK)), PDC1, xylose reductase (XR), xylitol dehydrogenase (XDH),L-(+)-lactate-cytochrome c oxidoreductase (CYB2), translation elongationfactor-1 (TEF1), translation elongation factor-2 (TEF2),glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and orotidine5′-phosphate decarboxylase (URA3) genes. Other suitable promoters may beobtained from S. cerevisiae TDH3, HXT7, PGK1, RPL18B and CCW12 genes.Additional useful promoters for yeast host cells are described byRomanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the yeast cell of choice may be used. The terminator maybe identical to or share a high degree of sequence identity (e.g., atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%) with the selected native terminator.

Suitable terminators for yeast host cells may be obtained from the genesfor enolase (e.g., S. cerevisiae or I. orientalis enolase cytochrome C(e.g., S. cerevisiae or I. orientalis cytochrome (CYC1)),glyceraldehyde-3-phosphate dehydrogenase (e.g., S. cerevisiae or I.orientalis glyceraldehyde-3-phosphate dehydrogenase (gpd)), PDC1, XR,XDH, transaldolase (TAL), transketolase (TKL), ribose 5-phosphateketol-isomerase (RKI), CYB2, and the galactose family of genes(especially the GAL10 terminator). Other suitable terminators may beobtained from S. cerevisiae ENO2 or TEF1 genes. Additional usefulterminators for yeast host cells are described by Romanos et al., 1992,supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis co/IIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the yeast cell of choice may beused.

Suitable leaders for yeast host cells are obtained from the genes forenolase (e.g., S. cerevisiae or I. orientalis enolase (ENO-1)),3-phosphoglycerate kinase (e.g., S. cerevisiae or I. orientalis3-phosphoglycerate kinase), alpha-factor (e.g., S. cerevisiae or I.orientalis alpha-factor), and alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (e.g., S.cerevisiae or I. orientalis alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP)).

The control sequence may also be a polyadenylation sequence; a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used. Usefulpolyadenylation sequences for yeast cells are described by Guo andSherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used.

The vectors may contain one or more (e.g., two, several) selectablemarkers that permit easy selection of transformed, transfected,transduced, or the like cells. A selectable marker is a gene the productof which provides for biocide or viral resistance, resistance to heavymetals, prototrophy to auxotrophs, and the like. Suitable markers foryeast host cells include, but are not limited to, ADE2, HIS3, LEU2,LYS2, MET3, TRP1, and URA3.

The vectors may contain one or more (e.g., two, several) elements thatpermit integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination. Potential integration loci include those described in theart (e.g., See US2012/0135481).

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the yeastcell. The origin of replication may be any plasmid replicator mediatingautonomous replication that functions in a cell. The term “origin ofreplication” or “plasmid replicator” means a polynucleotide that enablesa plasmid or vector to replicate in vivo. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6.

More than one copy of a polynucleotide described herein may be insertedinto a host cell to increase production of a polypeptide. An increase inthe copy number of the polynucleotide can be obtained by integrating atleast one additional copy of the sequence into the yeast cell genome orby including an amplifiable selectable marker gene with thepolynucleotide where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the polynucleotide, can beselected for by cultivating the cells in the presence of the appropriateselectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors described herein are well known toone skilled in the art (see, e.g., Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

Additional procedures and techniques known in the art for thepreparation of recombinant cells for ethanol fermentation, are describedin, e.g., WO 2016/045569, the content of which is hereby incorporated byreference.

The fermenting organism may be in the form of a composition comprising afermenting organism (e.g., a yeast strain described herein) and anaturally occurring and/or a nonenaturally occurring component.

The fermenting organism described herein may be in any viable form,including crumbled, dry, including active dry and instant, compressed,cream (liquid) form etc. In one embodiment, the fermenting organism(e.g., a Saccharomyces cerevisiae yeast strain) is dry yeast, such asactive dry yeast or instant yeast. In one embodiment, the fermentingorganism (e.g., a Saccharomyces cerevisiae yeast strain) is crumbledyeast. In one embodiment, the fermenting organism (e.g., a Saccharomycescerevisiae yeast strain) is compressed yeast. In one embodiment, thefermenting organism (e.g., a Saccharomyces cerevisiae yeast strain) iscream yeast.

In one embodiment is a composition comprising a fermenting organismdescribed herein (e.g., a Saccharomyces cerevisiae yeast strain), andone or more of the component selected from the group consisting of:surfactants, emulsifiers, gums, swelling agent, and antioxidants andother processing aids.

The compositions described herein may comprise a fermenting organismdescribed herein (e.g., a Saccharomyces cerevisiae yeast strain) and anysuitable surfactants. In one embodiment, the surfactant(s) is/are ananionic surfactant, cationic surfactant, and/or nonionic surfactant. Thecompositions described herein may comprise a fermenting organismdescribed herein (e.g., a Saccharomyces cerevisiae yeast strain) and anysuitable emulsifier. In one embodiment, the emulsifier is a fatty-acidester of sorbitan. In one embodiment, the emulsifier is selected fromthe group of sorbitan monostearate (SMS), citric acid esters ofmonodiglycerides, polyglycerolester, fatty acid esters of propyleneglycol.

In one embodiment, the composition comprises a fermenting organismdescribed herein (e.g., a Saccharomyces cerevisiae yeast strain), andOlindronal SMS, Olindronal SK, or Olindronal SPL including compositionconcerned in European Patent No. 1,724,336 (hereby incorporated byreference). These products are commercially available from Bussetti,Austria, for active dry yeast.

The compositions described herein may comprise a fermenting organismdescribed herein (e.g., a Saccharomyces cerevisiae yeast strain) and anysuitable gum. In one embodiment, the gum is selected from the group ofcarob, guar, tragacanth, arabic, xanthan and acacia gum, in particularfor cream, compressed and dry yeast.

The compositions described herein may comprise a fermenting organismdescribed herein (e.g., a Saccharomyces cerevisiae yeast strain) and anysuitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethyl cellulose.

The compositions described herein may comprise a fermenting organismdescribed herein (e.g., a Saccharomyces cerevisiae yeast strain) and anysuitable anti-oxidant. In one embodiment, the antioxidant is butylatedhydroxyanisol (BHA) and/or butylated hydroxytoluene (BHT), or ascorbicacid (vitamin C), particular for active dry yeast.

Gene Disruptions

The fermenting organisms described herein may also comprise one or more(e.g., two, several) gene disruptions, e.g., to divert sugar metabolismfrom undesired products to ethanol. In some aspects, the recombinanthost cells produce a greater amount of ethanol compared to the cellwithout the one or more disruptions when cultivated under identicalconditions. In some aspects, one or more of the disrupted endogenousgenes is inactivated.

In certain embodiments, the fermenting organism provided hereincomprises a disruption of one or more endogenous genes encoding enzymesinvolved in producing alternate fermentative products such as glycerolor other byproducts such as acetate or diols. For example, the cellsprovided herein may comprise a disruption of one or more of glycerol3-phosphate dehydrogenase (GPD, catalyzes reaction of dihydroxyacetonephosphate to glycerol 3-phosphate), glycerol 3-phosphatase (GPP,catalyzes conversion of glycerol-3 phosphate to glycerol), glycerolkinase (catalyzes conversion of glycerol 3-phosphate to glycerol),dihydroxyacetone kinase (catalyzes conversion of dihydroxyacetonephosphate to dihydroxyacetone), glycerol dehydrogenase (catalyzesconversion of dihydroxyacetone to glycerol), and aldehyde dehydrogenase(ALD, e.g., converts acetaldehyde to acetate).

Modeling analysis can be used to design gene disruptions thatadditionally optimize utilization of the pathway. One exemplarycomputational method for identifying and designing metabolic alterationsfavoring biosynthesis of a desired product is the OptKnock computationalframework, Burgard et al., 2003, Biotechnol. Bioeng. 84: 647-657.

The fermenting organisms comprising a gene disruption may be constructedusing methods well known in the art, including those methods describedherein. A portion of the gene can be disrupted such as the coding regionor a control sequence required for expression of the coding region. Sucha control sequence of the gene may be a promoter sequence or afunctional part thereof, i.e., a part that is sufficient for affectingexpression of the gene. For example, a promoter sequence may beinactivated resulting in no expression or a weaker promoter may besubstituted for the native promoter sequence to reduce expression of thecoding sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, propeptide sequence, signalsequence, transcription terminator, and transcriptional activator.

The fermenting organisms comprising a gene disruption may be constructedby gene deletion techniques to eliminate or reduce expression of thegene. Gene deletion techniques enable the partial or complete removal ofthe gene thereby eliminating their expression. In such methods, deletionof the gene is accomplished by homologous recombination using a plasmidthat has been constructed to contiguously contain the 5′ and 3′ regionsflanking the gene.

The fermenting organisms comprising a gene disruption may also beconstructed by introducing, substituting, and/or removing one or more(e.g., two, several) nucleotides in the gene or a control sequencethereof required for the transcription or translation thereof. Forexample, nucleotides may be inserted or removed for the introduction ofa stop codon, the removal of the start codon, or a frame-shift of theopen reading frame. Such a modification may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. See, for example, Botstein and Shortle,1985, Science 229: 4719; Lo et al., 1985, Proc. Natl. Acad. Sci. U.S.A.81: 2285; Higuchi et al., 1988, Nucleic Acids Res 16: 7351; Shimada,1996, Meth. Mol. Biol. 57: 157; Ho et al., 1989, Gene 77: 61; Horton etal., 1989, Gene 77: 61; and Sarkar and Sommer, 1990, BioTechniques 8:404.

The fermenting organisms comprising a gene disruption may also beconstructed by inserting into the gene a disruptive nucleic acidconstruct comprising a nucleic acid fragment homologous to the gene thatwill create a duplication of the region of homology and incorporateconstruct DNA between the duplicated regions. Such a gene disruption caneliminate gene expression if the inserted construct separates thepromoter of the gene from the coding region or interrupts the codingsequence such that a non-functional gene product results. A disruptingconstruct may be simply a selectable marker gene accompanied by 5′ and3′ regions homologous to the gene. The selectable marker enablesidentification of transformants containing the disrupted gene.

The fermenting organisms comprising a gene disruption may also beconstructed by the process of gene conversion (see, for example,Iglesias and Trautner, 1983, Molecular General Genetics 189: 73-76). Forexample, in the gene conversion method, a nucleotide sequencecorresponding to the gene is mutagenized in vitro to produce a defectivenucleotide sequence, which is then transformed into the recombinantstrain to produce a defective gene. By homologous recombination, thedefective nucleotide sequence replaces the endogenous gene. It may bedesirable that the defective nucleotide sequence also comprises a markerfor selection of transformants containing the defective gene.

The fermenting organisms comprising a gene disruption may be furtherconstructed by random or specific mutagenesis using methods well knownin the art, including, but not limited to, chemical mutagenesis (see,for example, Hopwood, The Isolation of Mutants in Methods inMicrobiology (J. R. Norris and D. W. Ribbons, eds.) pp. 363-433,Academic Press, New York, 1970). Modification of the gene may beperformed by subjecting the parent strain to mutagenesis and screeningfor mutant strains in which expression of the gene has been reduced orinactivated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, use of a suitable oligonucleotide, or subjecting theDNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesismay be performed by use of any combination of these mutagenizingmethods.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),N-methyl-N′-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid,ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, andnucleotide analogues. When such agents are used, the mutagenesis istypically performed by incubating the parent strain to be mutagenized inthe presence of the mutagenizing agent of choice under suitableconditions, and selecting for mutants exhibiting reduced or noexpression of the gene.

A nucleotide sequence homologous or complementary to a gene describedherein may be used from other microbial sources to disrupt thecorresponding gene in a recombinant strain of choice.

In one aspect, the modification of a gene in the recombinant cell isunmarked with a selectable marker. Removal of the selectable marker genemay be accomplished by culturing the mutants on a counter-selectionmedium. Where the selectable marker gene contains repeats flanking its5′ and 3′ ends, the repeats will facilitate the looping out of theselectable marker gene by homologous recombination when the mutantstrain is submitted to counter-selection. The selectable marker gene mayalso be removed by homologous recombination by introducing into themutant strain a nucleic acid fragment comprising 5′ and 3′ regions ofthe defective gene, but lacking the selectable marker gene, followed byselecting on the counter-selection medium. By homologous recombination,the defective gene containing the selectable marker gene is replacedwith the nucleic acid fragment lacking the selectable marker gene. Othermethods known in the art may also be used.

Methods using a Starch-Containing Material

In some aspects, the methods described herein produce a fermentationproduct from a starch-containing material. Starch-containing material iswell-known in the art, containing two types of homopolysaccharides(amylose and amylopectin) and is linked by alpha-(1-4)-D-glycosidicbonds. Any suitable starch-containing starting material may be used. Thestarting material is generally selected based on the desiredfermentation product, such as ethanol. Examples of starch-containingstarting materials include cereal, tubers or grains. Specifically, thestarch-containing material may be corn, wheat, barley, rye, milo, sago,cassava, tapioca, sorghum, oat, rice, peas, beans, or sweet potatoes, ormixtures thereof. Contemplated are also waxy and non-waxy types of cornand barley.

In one embodiment, the starch-containing starting material is corn. Inone embodiment, the starch-containing starting material is wheat. In oneembodiment, the starch-containing starting material is barley. In oneembodiment, the starch-containing starting material is rye. In oneembodiment, the starch-containing starting material is milo. In oneembodiment, the starch-containing starting material is sago. In oneembodiment, the starch-containing starting material is cassava. In oneembodiment, the starch-containing starting material is tapioca. In oneembodiment, the starch-containing starting material is sorghum. In oneembodiment, the starch-containing starting material is rice. In oneembodiment, the starch-containing starting material is peas. In oneembodiment, the starch-containing starting material is beans. In oneembodiment, the starch-containing starting material is sweet potatoes.In one embodiment, the starch-containing starting material is oats.

The methods using a starch-containing material may include aconventional process (e.g., including a liquefaction step described inmore detail below) or a raw starch hydrolysis process. In someembodiments using a starch-containing material, saccarification of thestarch-containing material is at a temperature above the initialgelatinization temperature. In some embodiments using astarch-containing material, saccarification of the starch-containingmaterial is at a temperature below the initial gelatinizationtemperature.

Liquefaction

In aspects using a starch-containing material, the methods may furthercomprise a liquefaction step carried out by subjecting thestarch-containing material at a temperature above the initialgelatinization temperature to an alpha-amylase and optionally a proteaseand/or a glucoamylase. Other enzymes such as a pullulanase and phytasemay also be present and/or added in liquefaction. In some embodiments,the liquefaction step is carried out prior to steps a) and b) of thedescribed methods.

Liquefaction step may be carried out for 0.5-5 hours, such as 1-3 hours,such as typically about 2 hours.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch-containing materialcommences. In general, starch heated in water begins to gelatinizebetween about 50° C. and 75° C.; the exact temperature of gelatinizationdepends on the specific starch and can readily be determined by theskilled artisan. Thus, the initial gelatinization temperature may varyaccording to the plant species, to the particular variety of the plantspecies as well as with the growth conditions. The initialgelatinization temperature of a given starch-containing material may bedetermined as the temperature at which birefringence is lost in 5% ofthe starch granules using the method described by Gorinstein and Lii,1992, Starch/Stärke 44(12): 461-466.

Liquefaction is typically carried out at a temperature in the range from70−100° C. In one embodiment, the temperature in liquefaction is between75-95° C., such as between 75-90° C., between 80-90° C., or between82-88° C., such as about 85° C.

A jet-cooking step may be carried out prior to liquefaction in step, forexample, at a temperature between 110-145° C., 120-140° C., 125-135° C.,or about 130° C. for about 1-15 minutes, for about 3-10 minutes, orabout 5 minutes.

The pH during liquefaction may be between 4 and 7, such as pH 4.5-6.5,pH 5.0-6.5, pH 5.0-6.0, pH 5.2-6.2, or about 5.2, about 5.4, about 5.6,or about 5.8.

In one embodiment, the process further comprises, prior to liquefaction,the steps of:

i) reducing the particle size of the starch-containing material,preferably by dry milling;

ii) forming a slurry comprising the starch-containing material andwater.

The starch-containing starting material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure, to increase surface area, and allowing for furtherprocessing. Generally, there are two types of processes: wet and drymilling. In dry milling whole kernels are milled and used. Wet millinggives a good separation of germ and meal (starch granules and protein).Wet milling is often applied at locations where the starch hydrolysateis used in production of, e.g., syrups. Both dry milling and wet millingare well known in the art of starch processing. In one embodiment thestarch-containing material is subjected to dry milling. In oneembodiment, the particle size is reduced to between 0.05 to 3.0 mm,e.g., 0.1-0.5 mm, or so that at least 30%, at least 50%, at least 70%,or at least 90% of the starch-containing material fit through a sievewith a 0.05 to 3.0 mm screen, e.g., 0.1-0.5 mm screen. In anotherembodiment, at least 50%, e.g., at least 70%, at least 80%, or at least90% of the starch-containing material fit through a sieve with #6screen.

The aqueous slurry may contain from 10-55 w/w-% dry solids (DS), e.g.,25-45 w/w-% dry solids (DS), or 30-40 w/w-% dry solids (DS) ofstarch-containing material.

The alpha-amylase, optionally a protease, and optionally a glucoamylasemay initially be added to the aqueous slurry to initiate liquefaction(thinning). In one embodiment, only a portion of the enzymes (e.g.,about ⅓) is added to the aqueous slurry, while the rest of the enzymes(e.g., about ⅔) are added during liquefaction step.

A non-exhaustive list of alpha-amylases used in liquefaction can befound below in the “Alpha-Amylases” section. Examples of suitableproteases used in liquefaction include any protease described supra inthe “Proteases” section. Examples of suitable glucoamylases used inliquefaction include any glucoamylase found in the “Glucoamylases”section.

Saccharification and Fermentation of Starch-Containing Material

In aspects using a starch-containing material, a glucoamylase may bepresent and/or added in saccharification step a) and/or fermentationstep b) or simultaneous saccharification and fermentation (SSF). Theglucoamylase of the saccharification step a) and/or fermentation step b)or simultaneous saccharification and fermentation (SSF) is typicallydifferent from the glucoamylase optionally added to any liquefactionstep described supra. In one embodiment, the glucoamylase is presentand/or added together with a fungal alpha-amylase.

In some aspects, the fermenting organism comprises a heterologouspolynucleotide encoding a glucoamylase, for example, as described inWO2017/087330, the content of which is hereby incorporated by reference.

Examples of glucoamylases can be found in the “Glucoamylases” sectionbelow.

When doing sequential saccharification and fermentation,saccharification step a) may be carried out under conditions well-knownin the art. For instance, saccharification step a) may last up to fromabout 24 to about 72 hours. In one embodiment, pre-saccharification isdone. Pre-saccharification is typically done for 40-90 minutes at atemperature between 30-65° C., typically about 60° C.Pre-saccharification is, in one embodiment, followed by saccharificationduring fermentation in simultaneous saccharification and fermentation(SSF). Saccharification is typically carried out at temperatures from20-75° C., preferably from 40-70° C., typically about 60° C., andtypically at a pH between 4 and 5, such as about pH 4.5.

Fermentation is carried out in a fermentation medium, as known in theart and, e.g., as described herein. The fermentation medium includes thefermentation substrate, that is, the carbohydrate source that ismetabolized by the fermenting organism. With the processes describedherein, the fermentation medium may comprise nutrients and growthstimulator(s) for the fermenting organism(s). Nutrient and growthstimulators are widely used in the art of fermentation and includenitrogen sources, such as ammonia; urea, vitamins and minerals, orcombinations thereof.

Generally, fermenting organisms such as yeast, including Saccharomycescerevisiae yeast, require an adequate source of nitrogen for propagationand fermentation. Many sources of supplemental nitrogen, if necessary,can be used and such sources of nitrogen are well known in the art. Thenitrogen source may be organic, such as urea, DDGs, wet cake or cornmash, or inorganic, such as ammonia or ammonium hydroxide. In oneembodiment, the nitrogen source is urea.

Fermentation can be carried out under low nitrogen conditions, e.g.,when using a protease-expressing yeast. In some embodiments, thefermentation step is conducted with less than 1000 ppm supplementalnitrogen (e.g., urea or ammonium hydroxide), such as less than 750 ppm,less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 250ppm, less than 200 ppm, less than 150 ppm, less than 100 ppm, less than75 ppm, less than 50 ppm, less than 25 ppm, or less than 10 ppm,supplemental nitrogen. In some embodiments, the fermentation step isconducted with no supplemental nitrogen.

Simultaneous saccharification and fermentation (“SSF”) is widely used inindustrial scale fermentation product production processes, especiallyethanol production processes. When doing SSF the saccharification stepa) and the fermentation step b) are carried out simultaneously. There isno holding stage for the saccharification, meaning that a fermentingorganism, such as yeast, and enzyme(s), may be added together. However,it is also contemplated to add the fermenting organism and enzyme(s)separately. SSF is typically carried out at a temperature from 25° C. to40° C., such as from 28° C. to 35° C., such as from 30° C. to 34° C., orabout 32° C. In one embodiment, fermentation is ongoing for 6 to 120hours, in particular 24 to 96 hours. In one embodiment, the pH isbetween 4-5.

In one embodiment, a cellulolytic enzyme composition is present and/oradded in saccharification, fermentation or simultaneous saccharificationand fermentation (SSF). Examples of such cellulolytic enzymecompositions can be found in the “Cellulolytic Enzymes and Compositions”section below. The cellulolytic enzyme composition may be present and/oradded together with a glucoamylase, such as one disclosed in the“Glucoamylases” section below.

Phospholipases

The expressed phospholipase may be any phospholipase that is suitablefor the host cells and/or the methods described herein, such as anaturally occurring phospholipase (e.g., a native phospholipase fromanother species or an endogenous phospholipase expressed from a modifiedexpression vector) or a variant thereof that retains phospholipaseactivity.

In some embodiments, the fermenting organism comprises a heterologouspolynucleotide encoding a phospholipase, for example, as described inWO2018/075430, the content of which is hereby incorporated by reference.In some embodiments, the phospholipase is classified as a phospholipaseA. In other embodiments, the phospholipase is classified as aphospholipase C.

Any phospholipase described or referenced herein is contemplated forexpression in the fermenting organism.

In some embodiments, the fermenting organism comprising a heterologouspolynucleotide encoding a phosphorlipase has an increased level ofphosphorlipase activity compared to the host cells without theheterologous polynucleotide encoding the phosphorlipase, when cultivatedunder the same conditions. In some embodiments, the fermenting organismhas an increased level of phospholipase activity of at least 5%, e.g.,at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, atleast 100%, at least 150%, at least 200%, at least 300%, or at 500%compared to the fermenting organism without the heterologouspolynucleotide encoding the phospholipase, when cultivated under thesame conditions.

Exemplary phospholipases that can be used with the host cells and/or themethods described herein include bacterial, yeast, or filamentous fungalphospholipases, e.g., derived from any of the microorganisms describedor referenced herein.

Additional phospholipases that may be expressed with the fermentingorganisms and used with the methods described herein are described inthe examples, and include, but are not limited to phospholipases shownin Table 1 (or derivatives thereof).

TABLE 1 Donor Organism SEQ ID NO: (catalytic domain) (maturepolypeptide) Thermomyces lanuginosus 235 Talaromyces leycettanus 236Penicillium emersonii 237 Bacillus thuringiensis 238 Pseudomonas sp. 239Kionochaeta sp. 240 Mariannaea pinicola 241 Fictibacillus macauensis 242Aspergillus wentii 252 Penicillium cylindrosporum 253 Penicilliummeridianum 254 Penicillium bialowiezense 255 Penicillium sclerotiorum256 Rasamsonia byssochlamydoides 257 Rasamsonia eburnea 258 Penicilliumbrefeldianum 259 Penicillium adametzii 260 Rasamsonia brevistipitata 261Penicillium scabrosum 262 Penicillium manginii 263 Penicillium emersonii264 Rasamsonia argillacea 265 Penicillium parviverrucosum 266Penicillium flavescens 267 Penicillium hispanicum 268 Penicilliumsimplicissimum 269 Penicillium vasconiae 270 Talaromyces columbinus 271Talaromyces variabilis 272 Talaromyces rugulosus 273 Hamigera terricola274 Penicillium piscarium 275 Talaromyces bacillisporus 276 Galactomycescandidus 277 Penicillium megasporum 278 Penicillium jensenii 279Aspergillus stramenius 280 Bacillus pseudomycoides 281 Bacillus mycoides282 Bacillus thuringiensis 283 Listeria innocua 284 Aspergillusegyptiacus 285 Aspergillus tamarii 286 Aspergillus niger 287 Bacillusluciferensis 288 Bacillus mycoides 289 Bacillus mycoides 290 Bacillussp. 291 Bacillus drentensis 292 Aspergillus turcosus 293 Talaromycessubinflatus 294 Aspergillus tubingensis 295 Bacillus acidiceler 296Lysinibacillus xylanilyticus 297 Bacillus toyonensis 298 Bacilluswiedmannii 299 Listeria seeligeri 300 Penicillium swiecickii 301Talaromyces boninensis 302 Hamigera striata 303 Bacillus sp. 304Bacillus thuringiensis 305 Bacillus mycoides 306 Fictibacillusmacauensis 307 Listeria seeligeri 308 Penicillium donkii 309 Hamigeraparavellanea 310 Talaromyces lecycettanus 311 Paenibacillus sp. 312Bacillus toyonensis 313 Bacillus thuringiensis 314 Bacillusthuringiensis 315 Talaromyces rugulosus 316 Penicillium sp. 317 Hamigeraavellanea 318 Penicillium spikei 319 Paenibacillus alginolyticus 320Bacillus mycoides 321 Bacillus bingmayongensis 322 Bacillus mycoides 323Brevibacillus sp. 324 Penicillium vasconiae 325 Talaromyces diversus 326Aspergillus wentii 327 Bacillus acidiceler 328 Bacillus luti 329Bacillus pseudomycoides 330 Bacillus mycoides 331 Penicilliumcinnamopurpureum 332 Talaromyces verruculosus 333 Talaromycescellulolyticus 334 Penicillium megasporum 335 Bacillus toyonensis 336Bacillus sp. 337 Bacillus manliponensis 338 Penicillium simplicissimum339 Penicillium arenicola 340 Aspergillus aculeatus 341 Bacillusacidiceler 342

Additional phospholipases contemplated for use with the presentinvention can be found in WO2018/075430 (the content of which isincorporated herein).

Additional polynucleotides encoding suitable phospholipases may beobtained from microorganisms of any genus, including those readilyavailable within the UniProtKB database (www.uniprot.org).

The phospholipase may be a bacterial phospholipase. For example, thephospholipase may be derived from a Gram-positive bacterium such as aBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, orStreptomyces, or a Gram-negative bacterium such as a Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, or Ureaplasma.

In one embodiment, the phospholipase is derived from Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus firmus,Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillusmegaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillussubtilis, or Bacillus thuringiensis.

In another embodiment, the phospholipase is derived from Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus.

In another embodiment, the phospholipase is derived from Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans.

The phospholipase may be a fungal phospholipase. For example, thephospholipase may be derived from a yeast such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia orIssatchenkia; or derived from a filamentous fungus such as anAcremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria.

In another embodiment, the phospholipase is derived from Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis.

In another embodiment, the phospholipase is derived from Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia austra/einsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The phospholipase coding sequences described or referenced herein, or asubsequence thereof, as well as the phospholipases described orreferenced herein, or a fragment thereof, may be used to design nucleicacid probes to identify and clone DNA encoding a phospholipase fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic DNA or cDNA of a cell of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, e.g., at least 25, atleast 35, or at least 70 nucleotides in length. Preferably, the nucleicacid probe is at least 100 nucleotides in length, e.g., at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 600 nucleotides, at least 700nucleotides, at least 800 nucleotides, or at least 900 nucleotides inlength. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin).

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith a coding sequence, or a subsequence thereof, the carrier materialis used in a Southern blot.

In one embodiment, the nucleic acid probe is a polynucleotide, orsubsequence thereof, that encodes the phospholipase of any one of SEQ IDNOs: 235-242 and 252-342 (such as the coding sequence of SEQ ID NOs:244-251 and 343-433, respectively), or a fragment thereof.

In one embodiment, the nucleic acid probe is a polynucleotide, orsubsequence thereof, that encodes the phospholipase of any one of SEQ IDNOs: 235, 236, 237, 238, 239, 240, 241 and 242 (such as the codingsequence of SEQ ID NO: 244, 245, 246, 247, 248, 249, 250 or 251,respectively), or a fragment thereof.

For purposes of the probes described above, hybridization indicates thatthe polynucleotide hybridizes to a labeled nucleic acid probe, or thefull-length complementary strand thereof, or a subsequence of theforegoing; under very low to very high stringency conditions. Moleculesto which the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film. Stringency and washingconditions are defined as described supra.

In one embodiment, the phospholipase is encoded by a polynucleotide thathybridizes under at least low stringency conditions, e.g., mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with thefull-length complementary strand of the coding sequence for any one ofthe phospholipases described or referenced herein (e.g., the codingsequence that encodes any one of SEQ ID NOs: 235-242 and 252-342; suchas the corresponding coding sequence of SEQ ID NO: 244-251 or 343-433,respectively, or the coding sequence that encodes any one of SEQ ID NOs:235, 236, 237, 238, 239, 240, 241 and 242; such as the correspondingcoding sequence of SEQ ID NO: 244, 245, 246, 247, 248, 249, 250 or 251,respectively). (Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.).

The phospholipase may also be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, silage, etc.) or DNA samples obtained directly from naturalmaterials (e.g., soil, composts, water, silage, etc.) using theabove-mentioned probes. Techniques for isolating microorganisms and DNAdirectly from natural habitats are well known in the art. Thepolynucleotide encoding a phospholipase may then be derived by similarlyscreening a genomic or cDNA library of another microorganism or mixedDNA sample.

Once a polynucleotide encoding a phospholipase has been detected with asuitable probe as described herein, the sequence may be isolated orcloned by utilizing techniques that are known to those of ordinary skillin the art (see, e.g., Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Techniquesused to isolate or clone polynucleotides encoding alpha-amylases includeisolation from genomic DNA, preparation from cDNA, or a combinationthereof. The cloning of the polynucleotides from such genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shares structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used.

In one embodiment, the phospholipase has a mature polypeptide sequencethat comprises or consists of the amino acid sequence of any one of thephospholipases described or referenced herein (e.g., any one of SEQ IDNOs: 235-242 and 252-342, such as any one of SEQ ID NOs: 235, 236, 237,238, 239, 240, 241 and 242). In another embodiment, the phospholipasehas a mature polypeptide sequence that is a fragment of the any one ofthe phospholipases described or referenced herein (e.g., any one of SEQID NOs: 235-242 and 252-342, such as any one of SEQ ID NOs: 235, 236,237, 238, 239, 240, 241 and 242). In one embodiment, the number of aminoacid residues in the fragment is at least 75%, e.g., at least 80%, 85%,90%, or 95% of the number of amino acid residues in referenced fulllength phospholipase (e.g., any one of SEQ ID NOs: 235-242 and 252-342,such as any one of SEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and242). In other embodiments, the phospholipase may comprise the catalyticdomain of any phospholipase described or referenced herein (e.g., thecatalytic domain of any one of SEQ ID NOs: 235-242 and 252-342, such asany one of SEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and 242).

The phospholipase may be a variant of any one of the phospholipasesdescribed supra (e.g., any one of SEQ ID NOs: 235-242 and 252-342, suchas any one of SEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and 242). Inone embodiment, the phospholipase has a mature polypeptide sequence ofat least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, 99%, or 100% sequence identity to any one of the phospholipasesdescribed supra (e.g., any one of SEQ ID NOs: 235-242 and 252-342, suchas any one of SEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and 242).

In one embodiment, the phospholipase has a mature polypeptide sequencethat differs by no more than ten amino acids, e.g., by no more than fiveamino acids, by no more than four amino acids, by no more than threeamino acids, by no more than two amino acids, or by one amino acid fromthe amino acid sequence of any one of the phospholipases described supra(e.g., any one of SEQ ID NOs: 235-242 and 252-342, such as any one ofSEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and 242). In oneembodiment, the phospholipase has an amino acid substitution, deletion,and/or insertion of one or more (e.g., two, several) of amino acidsequence of any one of the phospholipases described supra (e.g., any oneof SEQ ID NOs: 235-242 and 252-342, such as any one of SEQ ID NOs: 235,236, 237, 238, 239, 240, 241 and 242). In some embodiments, the totalnumber of amino acid substitutions, deletions and/or insertions is notmore than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1.

The amino acid changes are generally of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; smallamino-terminal or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thephospholipase, alter the substrate specificity, change the pH optimum,and the like.

Essential amino acids can be identified according to procedures known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, single alanine mutations are introduced at everyresidue in the molecule, and the resultant mutant molecules are testedfor activity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site or other biological interaction can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of identitieswith other phospholipases that are related to the referencedphospholipase.

Additional guidance on the structure-activity relationship of thepolypeptides herein can be determined using multiple sequence alignment(MSA) techniques well-known in the art. Based on the teachings herein,the skilled artisan could make similar alignments with any number ofphospholipases described herein or known in the art. Such alignments aidthe skilled artisan to determine potentially relevant domains (e.g.,binding domains or catalytic domains), as well as which amino acidresidues are conserved and not conserved among the differentalpha-amylase sequences. It is appreciated in the art that changing anamino acid that is conserved at a particular position between disclosedpolypeptides will more likely result in a change in biological activity(Bowie et al., 1990, Science 247: 1306-1310: “Residues that are directlyinvolved in protein functions such as binding or catalysis willcertainly be among the most conserved”). In contrast, substituting anamino acid that is not highly conserved among the polypeptides will notlikely or significantly alter the biological activity.

Even further guidance on the structure-activity relationship for theskilled artisan can be found in published x-ray crystallography studiesknown in the art.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activealpha-amylases can be recovered from the host cells and rapidlysequenced using standard methods in the art. These methods allow therapid determination of the importance of individual amino acid residuesin a polypeptide.

In some embodiments, the phospholipase has at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the phospholipase activity of any phospholipasedescribed or referenced herein (e.g., any one of SEQ ID NOs: 235-242 and252-342, such as any one of SEQ ID NOs: 235, 236, 237, 238, 239, 240,241 and 242) under the same conditions.

In one embodiment, the phospholipase coding sequence hybridizes under atleast low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any phospholipase described or referencedherein (e.g., a coding sequence for the phospholipase of any one of SEQID NOs: 235-242 and 252-342; such as the corresponding coding sequenceof SEQ ID NO: 244-251 or 343-433, respectively; or the phospholipase ofany one of SEQ ID NO: 235, 236, 237, 238, 239, 240, 241 or 242; such asthe corresponding coding sequence of SEQ ID NO: 244, 245, 246, 247, 248,249, 250 or 251, respectively). In one embodiment, the phospholipasecoding sequence has at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe coding sequence from any phospholipase described or referencedherein (e.g., a coding sequence for the phospholipase of any one of SEQID NOs: 235-242 and 252-342; such as the corresponding coding sequenceof SEQ ID NO: 244-251 or 343-433, respectively; or the phospholipase ofany one of SEQ ID NO: 235, 236, 237, 238, 239, 240, 241 or 242; such asthe corresponding coding sequence of SEQ ID NO: 244, 245, 246, 247, 248,249, 250 or 251, respectively).

In one embodiment, the phospholipase comprises the coding sequence ofany phospholipase described or referenced herein (e.g., a codingsequence for the phospholipase of any one of SEQ ID NOs: 235-242 and252-342; such as the corresponding coding sequence of SEQ ID NO: 244-251or 343-433, respectively; or the phospholipase of any one of SEQ ID NO:235, 236, 237, 238, 239, 240, 241 or 242; such as the correspondingcoding sequence of SEQ ID NO: 244, 245, 246, 247, 248, 249, 250 or 251,respectively). In one embodiment, the phospholipase comprises a codingsequence that is a subsequence of the coding sequence from anyphospholipase described or referenced herein, wherein the subsequenceencodes a polypeptide having phospholipase activity. In one embodiment,the number of nucleotides residues in the subsequence is at least 75%,e.g., at least 80%, 85%, 90%, or 95% of the number of the referencedcoding sequence.

The referenced coding sequence of any related aspect or embodimentdescribed herein can be the native coding sequence or a degeneratesequence, such as a codon-optimized coding sequence designed for use ina particular host cell (e.g., optimized for expression in Saccharomycescerevisiae).

The phospholipase may be a fused polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the alpha-amylase. A fused polypeptide may be producedby fusing a polynucleotide encoding another polypeptide to apolynucleotide encoding the phospholipase. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fused polypeptide is under control of the samepromoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

Alpha-Amylases

The expressed and/or exogenous alpha-amylase may be any alpha-amylasethat is suitable for the host cells and/or the methods described herein,such as a naturally occurring alpha-amylase (e.g., a nativealpha-amylase from another species or an endogenous alpha-amylaseexpressed from a modified expression vector) or a variant thereof thatretains alpha-amylase activity. Any alpha-amylase contemplated forexpression by a fermenting organism described below is also contemplatedfor aspects of the invention involving exogenous addition of analpha-amylase.

In some embodiments, the fermenting organism comprises a heterologouspolynucleotide encoding an alpha-amylase, for example, as described inWO2017/087330, the content of which is hereby incorporated by reference.Any alpha-amylase described or referenced herein is contemplated forexpression in the fermenting organism.

In some embodiments, the fermenting organism comprising a heterologouspolynucleotide encoding an alpha-amylase has an increased level ofalpha-amylase activity compared to the host cells without theheterologous polynucleotide encoding the alpha-amylase, when cultivatedunder the same conditions. In some embodiments, the fermenting organismhas an increased level of alpha-amylase activity of at least 5%, e.g.,at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, atleast 100%, at least 150%, at least 200%, at least 300%, or at 500%compared to the fermenting organism without the heterologouspolynucleotide encoding the alpha-amylase, when cultivated under thesame conditions.

Exemplary alpha-amylases that can be used with the host cells and/or themethods described herein include bacterial, yeast, or filamentous fungalalpha-amylases, e.g., derived from any of the microorganisms describedor referenced herein.

The term “bacterial alpha-amylase” means any bacterial alpha-amylaseclassified under EC 3.2.1.1. A bacterial alpha-amylase used herein may,e.g., be derived from a strain of the genus Bacillus, which is sometimesalso referred to as the genus Geobacillus. In one embodiment, theBacillus alpha-amylase is derived from a strain of Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus,or Bacillus subtilis, but may also be derived from other Bacillus sp.

Specific examples of bacterial alpha-amylases include the Bacillusstearothermophilus alpha-amylase (BSG) of SEQ ID NO: 3 in WO 99/19467,the Bacillus amyloliquefaciens alpha-amylase (BAN) of SEQ ID NO: 5 in WO99/19467, and the Bacillus licheniformis alpha-amylase (BLA) of SEQ IDNO: 4 in WO 99/19467 (all sequences are hereby incorporated byreference). In one embodiment, the alpha-amylase may be an enzyme havinga mature polypeptide sequence with a degree of identity of at least 60%,e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% to any of the sequencesshown in SEQ ID NOs: 3, 4 or 5, in WO 99/19467.

In one embodiment, the alpha-amylase is derived from Bacillusstearothermophilus. The Bacillus stearothermophilus alpha-amylase may bea mature wild-type or a mature variant thereof. The mature Bacillusstearothermophilus alpha-amylases may naturally be truncated duringrecombinant production. For instance, the Bacillus stearothermophilusalpha-amylase may be a truncated at the C-terminal, so that it is from480-495 amino acids long, such as about 491 amino acids long, e.g., sothat it lacks a functional starch binding domain (compared to SEQ ID NO:3 in WO 99/19467).

The Bacillus alpha-amylase may also be a variant and/or hybrid. Examplesof such a variant can be found in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (each herebyincorporated by reference). Specific alpha-amylase variants aredisclosed in U.S. Pat. Nos. 6,093,562, 6,187,576, 6,297,038, and7,713,723 (hereby incorporated by reference) and include Bacillusstearothermophilus alpha-amylase (often referred to as BSGalpha-amylase) variants having a deletion of one or two amino acids atpositions R179, G180, 1181 and/or G182, preferably a double deletiondisclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (herebyincorporated by reference), such as corresponding to deletion ofpositions 1181 and G182 compared to the amino acid sequence of Bacillusstearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed inWO 99/19467 or the deletion of amino acids R179 and G180 using SEQ IDNO: 3 in WO 99/19467 for numbering (which reference is herebyincorporated by reference). In some embodiments, the Bacillusalpha-amylases, such as Bacillus stearothermophilus alpha-amylases, havea double deletion corresponding to a deletion of positions 181 and 182and further optionally comprise a N193F substitution (also denoted1181*+G182*+N193F) compared to the wild-type BSG alpha-amylase aminoacid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467. Thebacterial alpha-amylase may also have a substitution in a positioncorresponding to S239 in the Bacillus licheniformis alpha-amylase shownin SEQ ID NO: 4 in WO 99/19467, or a S242 and/or E188P variant of theBacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO99/19467. In one embodiment, the variant is a S242A, E or Q variant,e.g., a S242Q variant, of the Bacillus stearothermophilus alpha-amylase.

In one embodiment, the variant is a position E188 variant, e.g., E188Pvariant of the Bacillus stearothermophilus alpha-amylase.

The bacterial alpha-amylase may, in one embodiment, be a truncatedBacillus alpha-amylase. In one embodiment, the truncation is so that,e.g., the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO:3 in WO 99/19467, is about 491 amino acids long, such as from 480 to 495amino acids long, or so it lacks a functional starch bind domain.

The bacterial alpha-amylase may also be a hybrid bacterialalpha-amylase, e.g., an alpha-amylase comprising 445 C-terminal aminoacid residues of the Bacillus licheniformis alpha-amylase (shown in SEQID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues ofthe alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQID NO: 5 of WO 99/19467). In one embodiment, this hybrid has one ormore, especially all, of the following substitutions:G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the Bacilluslicheniformis numbering in SEQ ID NO: 4 of WO 99/19467). In someembodiments, the variants have one or more of the following mutations(or corresponding mutations in other Bacillus alpha-amylases): H154Y,A181T, N190F, A209V and Q264S and/or the deletion of two residuesbetween positions 176 and 179, e.g., deletion of E178 and G179 (usingSEQ ID NO: 5 of WO 99/19467 for position numbering).

In one embodiment, the bacterial alpha-amylase is the mature part of thechimeric alpha-amylase disclosed in Richardson et al. (2002), TheJournal of Biological Chemistry, Vol. 277, No 29, Issue 19 July, pp.267501-26507, referred to as BD5088 or a variant thereof. Thisalpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO2007/134207. The mature enzyme sequence starts after the initial “Met”amino acid in position 1.

The alpha-amylase may be a thermostable alpha-amylase, such as athermostable bacterial alpha-amylase, e.g., from Bacillusstearothermophilus. In one embodiment, the alpha-amylase used in aprocess described herein has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂of at least 10 determined as described in Example 1 of WO2018/098381.

In one embodiment, the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of at least 15. In one embodiment, thethermostable alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mMCaCl₂, of as at least 20. In one embodiment, the thermostablealpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂, of as atleast 25. In one embodiment, the thermostable alpha-amylase has a T½(min) at pH 4.5, 85° C., 0.12 mM CaCl₂, of as at least 30. In oneembodiment, the thermostable alpha-amylase has a T½ (min) at pH 4.5, 85°C., 0.12 mM CaCl₂, of as at least 40.

In one embodiment, the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of at least 50. In one embodiment, thethermostable alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mMCaCl₂, of at least 60. In one embodiment, the thermostable alpha-amylasehas a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂, between 10-70. In oneembodiment, the thermostable alpha-amylase has a T½ (min) at pH 4.5, 85°C., 0.12 mM CaCl₂, between 15-70. In one embodiment, the thermostablealpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂, between20-70. In one embodiment, the thermostable alpha-amylase has a T½ (min)at pH 4.5, 85° C., 0.12 mM CaCl₂, between 25-70. In one embodiment, thethermostable alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mMCaCl₂, between 30-70. In one embodiment, the thermostable alpha-amylasehas a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂, between 40-70. In oneembodiment, the thermostable alpha-amylase has a T½ (min) at pH 4.5, 85°C., 0.12 mM CaCl₂, between 50-70. In one embodiment, the thermostablealpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂, between60-70.

In one embodiment, the alpha-amylase is a bacterial alpha-amylase, e.g.,derived from the genus Bacillus, such as a strain of Bacillusstearothermophilus, e.g., the Bacillus stearothermophilus as disclosedin WO 99/019467 as SEQ ID NO: 3 with one or two amino acids deleted atpositions R179, G180, 1181 and/or G182, in particular with R179 and G180deleted, or with 1181 and G182 deleted, with mutations in below list ofmutations.

In some embodiment, the Bacillus stearothermophilus alpha-amylases havedouble deletion 1181+G182, and optional substitution N193F, furthercomprising one of the following substitutions or combinations ofsubstitutions:

V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;

V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+1270L;

V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;

V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;

V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;

V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;

V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;

V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;

V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;

V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;

V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;

V59A+E129V+K177L+R179E+K220P+N224L+Q254S;

V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;

A91L+M961+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

E129V+K177L+R179E;

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+1377*;

E129V+K177L+R179E+K220P+N224L+Q254S;

E129V+K177L+R179E+K220P+N224L+Q254S+M284T;

E129V+K177L+R179E+S242Q;

E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;

K220P+N224L+S242Q+Q254S;

M284V;

V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; and

V59A+E129V+K177L+R179E+Q254S+M284V;

In one embodiment, the alpha-amylase is selected from the group ofBacillus stearothermophilus alpha-amylase variants with double deletion1181*+G182*, and optionally substitution N193F, and further one of thefollowing substitutions or combinations of substitutions:

E129V+K177L+R179E;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;

V59A+E129V+K177L+R179E+Q254S+M284V; and

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1 herein fornumbering).

It should be understood that when referring to Bacillusstearothermophilus alpha-amylase and variants thereof they are normallyproduced in truncated form. In particular, the truncation may be so thatthe Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 inWO 99/19467, or variants thereof, are truncated in the C-terminal andare typically from 480-495 amino acids long, such as about 491 aminoacids long, e.g., so that it lacks a functional starch binding domain.

In one embodiment, the alpha-amylase variant may be an enzyme having amature polypeptide sequence with a degree of identity of at least 60%,e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99%, but less than 100% tothe sequence shown in SEQ ID NO: 3 in WO 99/19467.

In one embodiment, the bacterial alpha-amylase, e.g., Bacillusalpha-amylase, such as especially Bacillus stearothermophilusalpha-amylase, or variant thereof, is dosed to liquefaction in aconcentration between 0.01-10 KNU-A/g DS, e.g., between 0.02 and 5KNU-A/g DS, such as 0.03 and 3 KNU-A, preferably 0.04 and 2 KNU-A/g DS,such as especially 0.01 and 2 KNU-A/g DS. In one embodiment, thebacterial alpha-amylase, e.g., Bacillus alpha-amylase, such asespecially Bacillus stearothermophilus alpha-amylases, or variantthereof, is dosed to liquefaction in a concentration of between 0.0001-1mg EP (Enzyme Protein)/g DS, e.g., 0.0005-0.5 mg EP/g DS, such as0.001-0.1 mg EP/g DS.

In one embodiment, the bacterial alpha-amylase is derived from theBacillus subtilis alpha-amylase of SEQ ID NO: 76, the Bacillus subtilisalpha-amylase of SEQ ID NO: 82, the Bacillus subtilis alpha-amylase ofSEQ ID NO: 83, the Bacillus subtilis alpha-amylase of SEQ ID NO: 84, orthe Bacillus licheniformis alpha-amylase of SEQ ID NO: 85, theClostridium phytofermentans alpha-amylase of SEQ ID NO: 89, theClostridium phytofermentans alpha-amylase of SEQ ID NO: 90, theClostridium phytofermentans alpha-amylase of SEQ ID NO: 91, theClostridium phytofermentans alpha-amylase of SEQ ID NO: 92, theClostridium phytofermentans alpha-amylase of SEQ ID NO: 93, theClostridium phytofermentans alpha-amylase of SEQ ID NO: 94, theClostridium thermocellum alpha-amylase of SEQ ID NO: 95, theThermobifida fusca alpha-amylase of SEQ ID NO: 96, the Thermobifidafusca alpha-amylase of SEQ ID NO: 97, the Anaerocellum thermophilum ofSEQ ID NO: 98, the Anaerocellum thermophilum of SEQ ID NO: 99, theAnaerocellum thermophilum of SEQ ID NO: 100, the Streptomycesavermitilis of SEQ ID NO: 101, or the Streptomyces avermitilis of SEQ IDNO: 88.

In one embodiment, the alpha-amylase is derived from Bacillusamyloliquefaciens, such as the Bacillus amyloliquefaciens alpha-amylaseof SEQ ID NO: 231 (e.g., as described in WO2018/002360, or variantsthereof as described in WO2017/037614).

In one embodiment, the alpha-amylase is derived from a yeastalpha-amylase, such as the Saccharomycopsis fibuligera alpha-amylase ofSEQ ID NO: 77, the Debaryomyces occidentalis alpha-amylase of SEQ ID NO:78, the Debaryomyces occidentalis alpha-amylase of SEQ ID NO: 79, theLipomyces kononenkoae alpha-amylase of SEQ ID NO: 80, the Lipomyceskononenkoae alpha-amylase of SEQ ID NO: 81.

In one embodiment, the alpha-amylase is derived from a filamentousfungal alpha-amylase, such as the Aspergillus niger alpha-amylase of SEQID NO: 86, or the Aspergillus niger alpha-amylase of SEQ ID NO: 87.

Additional alpha-amylases that may be expressed with the fermentingorganisms and used with the methods described herein are described inthe examples, and include, but are not limited to alpha-amylases shownin Table 2 (or derivatives thereof).

TABLE 2 Donor Organism SEQ ID NO: (catalytic domain) (maturepolypeptide) Rhizomucor pusillus 121 Bacillus licheniformis 122Aspergillus niger 123 Aspergillus tamarii 124 Acidomyces richmondensis125 Aspergillus bombycis 126 Alternaria sp 127 Rhizopus microsporus 128Syncephalastrum racemosum 129 Rhizomucor pusillus 130 Dichotomocladiumhesseltinei 131 Lichtheimia ramosa 132 Penicillium aethiopicum 133Subulispora sp 134 Trichoderma paraviridescens 135 Byssoascusstriatosporus 136 Aspergillus brasiliensis 137 Penicillium subspinulosum138 Penicillium antarcticum 139 Penicillium coprophilum 140 Penicilliumolsonii 141 Penicillium vasconiae 142 Penicillium sp 143 Heterocephalumaurantiacum 144 Neosartorya massa 145 Penicillium janthinellum 146Aspergillus brasiliensis 147 Aspergillus westerdijkiae 148 Hamigeraavellanea 149 Hamigera avellanea 150 Meripilus giganteus 151 Cerrenaunicolor 152 Physalacria cryptomeriae 153 Lenzites betulinus 154Trametes ljubarskyi 155 Bacillus subtilis 156 Bacillus subtilis subsp.subtilis 157 Schwanniomyces occidentalis 158 Rhizomucor pusillus 159Aspergillus niger 160 Bacillus stearothermophilus 161 Bacillushalmapalus 162 Aspergillus oryzae 163 Bacillus amyloliquefaciens 164Rhizomucor pusillus 165 Kionochaeta ivoriensis 166 Aspergillus niger 167Aspergillus oryzae 168 Penicillium canescens 169 Acidomyces acidothermus170 Kinochaeta ivoriensis 171 Aspergillus terreus 172 Thamnidium elegans173 Meripilus giganteus 174

Additional alpha-amylases contemplated for use with the presentinvention can be found in WO2011/153516 (the content of which isincorporated herein).

Additional polynucleotides encoding suitable alpha-amylases may beobtained from microorganisms of any genus, including those readilyavailable within the UniProtKB database (www.uniprot.org).

The alpha-amylase coding sequences can also be used to design nucleicacid probes to identify and clone DNA encoding trehalases from strainsof different genera or species, as described supra.

The polynucleotides encoding alpha-amylases may also be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encodingalpha-amylases are described supra.

In one embodiment, the alpha-amylase has a mature polypeptide sequencethat comprises or consists of the amino acid sequence of any one of thealpha-amylases described or referenced herein (e.g., any one of SEQ IDNOs: 76-101, 121-174 and 231). In another embodiment, the alpha-amylasehas a mature polypeptide sequence that is a fragment of the any one ofthe alpha-amylases described or referenced herein (e.g., any one of SEQID NOs: 76-101, 121-174 and 231). In one embodiment, the number of aminoacid residues in the fragment is at least 75%, e.g., at least 80%, 85%,90%, or 95% of the number of amino acid residues in referenced fulllength alpha-amylase (e.g. any one of SEQ ID NOs: 76-101, 121-174 and231). In other embodiments, the alpha-amylase may comprise the catalyticdomain of any alpha-amylase described or referenced herein (e.g., thecatalytic domain of any one of SEQ ID NOs: 76-101, 121-174 and 231).

The alpha-amylase may be a variant of any one of the alpha-amylasesdescribed supra (e.g., any one of SEQ ID NOs: 76-101, 121-174 and 231).In one embodiment, the alpha-amylase has a mature polypeptide sequenceof at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, 99%, or 100% sequence identity to any one of the alpha-amylasesdescribed supra (e.g., any one of SEQ ID NOs: 76-101, 121-174 and 231).

In one embodiment, the alpha-amylase has a mature polypeptide sequencethat differs by no more than ten amino acids, e.g., by no more than fiveamino acids, by no more than four amino acids, by no more than threeamino acids, by no more than two amino acids, or by one amino acid fromthe amino acid sequence of any one of the alpha-amylases described supra(e.g., any one of SEQ ID NOs: 76-101, 121-174 and 231). In oneembodiment, the alpha-amylase has an amino acid substitution, deletion,and/or insertion of one or more (e.g., two, several) of amino acidsequence of any one of the alpha-amylases described supra (e.g., any oneof SEQ ID NOs: 76-101, 121-174 and 231). In some embodiments, the totalnumber of amino acid substitutions, deletions and/or insertions is notmore than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1.

In some embodiments, the alpha-amylase has at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the alpha-amylase activity of any alpha-amylasedescribed or referenced herein (e.g., any one of SEQ ID NOs: 76-101,121-174 and 231) under the same conditions.

In one embodiment, the alpha-amylase coding sequence hybridizes under atleast low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any alpha-amylase described or referencedherein (e.g., any one of SEQ ID NOs: 76-101, 121-174 and 231). In oneembodiment, the alpha-amylase coding sequence has at least 65%, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the coding sequence from any alpha-amylasedescribed or referenced herein (e.g., any one of SEQ ID NOs: 76-101,121-174 and 231).

In one embodiment, the alpha-amylase comprises the coding sequence ofany alpha-amylase described or referenced herein (any one of SEQ ID NOs:76-101, 121-174 and 231). In one embodiment, the alpha-amylase comprisesa coding sequence that is a subsequence of the coding sequence from anyalpha-amylase described or referenced herein, wherein the subsequenceencodes a polypeptide having alpha-amylase activity. In one embodiment,the number of nucleotides residues in the subsequence is at least 75%,e.g., at least 80%, 85%, 90%, or 95% of the number of the referencedcoding sequence.

The referenced coding sequence of any related aspect or embodimentdescribed herein can be the native coding sequence or a degeneratesequence, such as a codon-optimized coding sequence designed for use ina particular host cell (e.g., optimized for expression in Saccharomycescerevisiae).

The alpha-amylase can also include fused polypeptides or cleavablefusion polypeptides, as described supra.

Trehalases

The expressed and/or exogenous trehalase can be any trehalase that issuitable for the fermenting organisms and/or their methods of usedescribed herein, such as a naturally occurring trehalase or a variantthereof that retains trehalase activity. Any trehalase contemplated forexpression by a fermenting organism described below is also contemplatedfor aspects of the invention involving exogenous addition of a trehalase(e.g., added before, during or after liquefaction and/orsaccharification).

In some embodiments, the fermenting organism comprising a heterologouspolynucleotide encoding a trehalase has an increased level of trehalaseactivity compared to the host cells without the heterologouspolynucleotide encoding the trehalase, when cultivated under the sameconditions. In some embodiments, the fermenting organism has anincreased level of trehalase activity of at least 5%, e.g., at least10%, at least 15%, at least 20%, at least 25%, at least 50%, at least100%, at least 150%, at least 200%, at least 300%, or at 500% comparedto the fermenting organism without the heterologous polynucleotideencoding the trehalase, when cultivated under the same conditions.

Trehalases that may be expressed with the fermenting organisms and usedwith the methods described herein include, but are not limited to,trehalases shown in Table 3 (or derivatives thereof).

TABLE 3 Donor Organism SEQ ID NO: (catalytic domain) (maturepolypeptide) Chaetomium megalocarpum 175 Lecanicillium psalliotae 176Doratomyces sp 177 Mucor moelleri 178 Phialophora cyclaminis 179Thielavia arenaria 180 Thielavia antarctica 181 Chaetomium sp 182Chaetomium nigricolor 183 Chaetomium jodhpurense 184 Chaetomiumpiluliferum 185 Myceliophthora hinnulea 186 Chloridium virescens 187Gelasinospora cratophora 188 Acidobacteriaceae bacterium 189Acidobacterium capsulatum 190 Acidovorax wautersii 191 Xanthomonasarborícola 192 Kosakonia sacchari 193 Enterobacter sp 194 Saitozymaflava 195 Phaeotremella skinneri 196 Trichoderma asperellum 197Corynascus sepedonium 198 Myceliophthora thermophila 199 Trichodermareesei 200 Chaetomium virescens 201 Rhodothermus marinus 202Myceliophthora sepedonium 203 Moelleriella libera 204 Acremoniumdichromosporum 205 Fusarium sambucinum 206 Phoma sp 207 Lentinus similis208 Diaporthe nobilis 209 Solicoccozyma terricola 210 Dioszegiacryoxerica 211 Talaromyces funiculosus 212 Hamigera avellanea 213Talaromyces ruber 214 Trichoderma lixii 215 Aspergillus cervinus 216Rasamsonia brevistipitata 217 Acremonium curvulum 218 Talaromyces piceae219 Penicillium sp 220 Talaromyces aurantiacus 221 Talaromycespinophilus 222 Talaromyces leycettanus 223 Talaromyces variabilis 224Aspergillus niger 225 Trichoderma reesei 226

Additional polynucleotides encoding suitable trehalases may be derivedfrom microorganisms of any suitable genus, including those readilyavailable within the UniProtKB database (www.uniprot.org).

The trehalase coding sequences can also be used to design nucleic acidprobes to identify and clone DNA encoding trehalases from strains ofdifferent genera or species, as described supra.

The polynucleotides encoding trehalases may also be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encoding trehalasesare described supra.

In one embodiment, the trehalase has a mature polypeptide sequence thatcomprises or consists of the amino acid sequence of any one of thetrehalases described or referenced herein (e.g., any one of SEQ ID NOs:175-226). In another embodiment, the trehalase has a mature polypeptidesequence that is a fragment of the any one of the trehalases describedor referenced herein (e.g., any one of SEQ ID NOs: 175-226). In oneembodiment, the number of amino acid residues in the fragment is atleast 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of aminoacid residues in referenced full length trehalase (e.g. any one of SEQID NOs: 175-226). In other embodiments, the trehalase may comprise thecatalytic domain of any trehalase described or referenced herein (e.g.,the catalytic domain of any one of SEQ ID NOs: 175-226).

The trehalase may be a variant of any one of the trehalases describedsupra (e.g., any one of SEQ ID NOs: 175-226). In one embodiment, thetrehalase has a mature polypeptide sequence of at least 60%, e.g., atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity to any one of the trehalases described supra (e.g., any one ofSEQ ID NOs: 175-226).

In one embodiment, the trehalase has a mature polypeptide sequence thatdiffers by no more than ten amino acids, e.g., by no more than fiveamino acids, by no more than four amino acids, by no more than threeamino acids, by no more than two amino acids, or by one amino acid fromthe amino acid sequence of any one of the trehalases described supra(e.g., any one of SEQ ID NOs: 175-226). In one embodiment, the trehalasehas an amino acid substitution, deletion, and/or insertion of one ormore (e.g., two, several) of amino acid sequence of any one of thetrehalases described supra (e.g., any one of SEQ ID NOs: 175-226). Insome embodiments, the total number of amino acid substitutions,deletions and/or insertions is not more than 10, e.g., not more than 9,8, 7, 6, 5, 4, 3, 2, or 1.

In some embodiments, the trehalase has at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% of the trehalase activity of any trehalase described or referencedherein (e.g., any one of SEQ ID NOs: 175-226) under the same conditions.

In one embodiment, the trehalase coding sequence hybridizes under atleast low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any trehalase described or referenced herein(e.g., any one of SEQ ID NOs: 175-226). In one embodiment, the trehalasecoding sequence has at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe coding sequence from any trehalase described or referenced herein(e.g., any one of SEQ ID NOs: 175-226).

In one embodiment, the trehalase comprises the coding sequence of anytrehalase described or referenced herein (any one of SEQ ID NOs:175-226). In one embodiment, the trehalase comprises a coding sequencethat is a subsequence of the coding sequence from any trehalasedescribed or referenced herein, wherein the subsequence encodes apolypeptide having trehalase activity. In one embodiment, the number ofnucleotides residues in the subsequence is at least 75%, e.g., at least80%, 85%, 90%, or 95% of the number of the referenced coding sequence.

The referenced coding sequence of any related aspect or embodimentdescribed herein can be the native coding sequence or a degeneratesequence, such as a codon-optimized coding sequence designed for use ina particular host cell (e.g., optimized for expression in Saccharomycescerevisiae).

The trehalase can also include fused polypeptides or cleavable fusionpolypeptides, as described supra.

Glucoamylases

The expressed and/or exogenous glucoamylase can be any glucoamylase thatis suitable for the fermenting organisms and/or their methods of usedescribed herein, such as a naturally occurring glucoamylase or avariant thereof that retains glucoamylase activity. Any glucoamylasecontemplated for expression by a fermenting organism described below isalso contemplated for aspects of the invention involving exogenousaddition of a glucoamylase (e.g., added before, during or afterliquefaction and/or saccharification).

In some embodiments, the fermenting organism comprises a heterologouspolynucleotide encoding a glucoamylase, for example, as described inWO2017/087330, the content of which is hereby incorporated by reference.Any glucoamylase described or referenced herein is contemplated forexpression in the fermenting organism.

In some embodiments, the fermenting organism comprising a heterologouspolynucleotide encoding an glucoamylase has an increased level ofglucoamylase activity compared to the host cells without theheterologous polynucleotide encoding the glucoamylase, when cultivatedunder the same conditions. In some embodiments, the fermenting organismhas an increased level of glucoamylase activity of at least 5%, e.g., atleast 10%, at least 15%, at least 20%, at least 25%, at least 50%, atleast 100%, at least 150%, at least 200%, at least 300%, or at 500%compared to the fermenting organism without the heterologouspolynucleotide encoding the glucoamylase, when cultivated under the sameconditions.

Exemplary glucoamylases that can be used with the host cells and/or themethods described herein include bacterial, yeast, or filamentous fungalglucoamylases, e.g., obtained from any of the microorganisms describedor referenced herein, as described supra under the sections related toalpha-amylases.

The glucoamylase may be derived from any suitable source, e.g., derivedfrom a microorganism or a plant. Preferred glucoamylases are of fungalor bacterial origin, selected from the group consisting of Aspergillusglucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase(Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof,such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273(from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO84/02921, Aspergillus oryzae glucoamylase (Agric. Biol. Chem. (1991), 55(4), p. 941-949), or variants or fragments thereof. Other Aspergillusglucoamylase variants include variants with enhanced thermal stability:G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E andD293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al.(1994), Biochem. J. 301, 275-281); disulphide bonds, A246C (Fierobe etal. (1996), Biochemistry, 35, 8698-8704; and introduction of Proresidues in position A435 and S436 (Li et al. (1997), Protein Eng. 10,1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka et al. (1998) “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat No. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215). In one embodiment, theglucoamylase used during saccharification and/or fermentation is theTalaromyces emersonii glucoamylase disclosed in WO 99/28448.

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831).

Contemplated fungal glucoamylases include Trametes cingulate,Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed inWO 2006/069289; or Peniophora rufomarginata disclosed in WO2007/124285;or a mixture thereof. Also hybrid glucoamylase are contemplated.Examples include the hybrid glucoamylases disclosed in WO 2005/045018.

In one embodiment, the glucoamylase is derived from a strain of thegenus Pycnoporus, in particular a strain of Pycnoporus as described inWO 2011/066576 (SEQ ID NO: 2, 4 or 6 therein), including the Pycnoporussanguineus glucoamylase, or from a strain of the genus Gloeophyllum,such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, inparticular a strain of Gloeophyllum as described in WO 2011/068803 (SEQID NO: 2, 4, 6, 8, 10, 12, 14 or 16 therein). In one embodiment, theglucoamylase is SEQ ID NO: 2 in WO 2011/068803 (i.e. Gloeophyllumsepiarium glucoamylase). In one embodiment, the glucoamylase is theGloeophyllum sepiarium glucoamylase of SEQ ID NO: 8. In one embodiment,the glucoamylase is the Pycnoporus sanguineus glucoamylase of SEQ ID NO:229.

In one embodiment, the glucoamylase is a Gloeophyllum trabeumglucoamylase (disclosed as SEQ ID NO: 3 in WO2014/177546). In anotherembodiment, the glucoamylase is derived from a strain of the genusNigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO2012/064351 (disclosed as SEQ ID NO: 2 therein).

Also contemplated are glucoamylases with a mature polypeptide sequencewhich exhibit a high identity to any of the above mentionedglucoamylases, i.e., at least 60%, such as at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or even 100% identity to any oneof the mature polypeptide sequences mentioned above.

Glucoamylases may be added to the saccharification and/or fermentationin an amount of 0.0001-20 AGU/g DS, such as 0.001-10 AGU/g DS, 0.01-5AGU/g DS, or 0.1-2 AGU/g DS.

Glucoamylases may be added to the saccharification and/or fermentationin an amount of 1-1,000 μg EP/g DS, such as 10-500 μg/gDS, or 25-250μg/g DS.

Glucoamylases may be added to liquefaction in an amount of 0.1-100 μgEP/g DS, such as 0.5-50 μg EP/g DS, 1-25 μg EP/g DS, or 2-12 μg EP/g DS.

In one embodiment, the glucoamylase is added as a blend furthercomprising an alpha-amylase (e.g., any alpha-amylase described herein).In one embodiment, the alpha-amylase is a fungal alpha-amylase,especially an acid fungal alpha-amylase. The alpha-amylase is typicallya side activity.

In one embodiment, the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO 99/28448 as SEQ ID NO: 34 andTrametes cingulata glucoamylase disclosed as SEQ ID NO: 2 in WO06/069289.

In one embodiment, the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO 99/28448, Trametes cingulataglucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289, and analpha-amylase.

In one embodiment, the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO99/28448, Trametes cingulataglucoamylase disclosed in WO 06/69289, and Rhizomucor pusillusalpha-amylase with Aspergillus niger glucoamylase linker and SBDdisclosed as V039 in Table 5 in WO 2006/069290.

In one embodiment, the glucoamylase is a blend comprising Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 2 in WO 2011/068803 and analpha-amylase, in particular Rhizomucor pusillus alpha-amylase with anAspergillus niger glucoamylase linker and starch-binding domain (SBD),disclosed SEQ ID NO: 3 in WO 2013/006756, in particular with thefollowing substitutions: G128D+D143N.

In one embodiment, the alpha-amylase may be derived from a strain of thegenus Rhizomucor, preferably a strain the Rhizomucor pusillus, such asthe one shown in SEQ ID NO: 3 in WO2013/006756, or the genus Meripilus,preferably a strain of Meripilus giganteus. In one embodiment, thealpha-amylase is derived from a Rhizomucor pusillus with an Aspergillusniger glucoamylase linker and starch-binding domain (SBD), disclosed asV039 in Table 5 in WO 2006/069290.

In one embodiment, the Rhizomucor pusillus alpha-amylase or theRhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylaselinker and starch-binding domain (SBD) has at least one of the followingsubstitutions or combinations of substitutions: D165M; Y141W; Y141R;K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W;G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R;Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N;Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C;Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; andG128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ IDNO: 3 in WO 2013/006756 for numbering).

In one embodiment, the glucoamylase blend comprises Gloeophyllumsepiarium glucoamylase (e.g., SEQ ID NO: 2 in WO 2011/068803) andRhizomucor pusillus alpha-amylase.

In one embodiment, the glucoamylase blend comprises Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 2 in WO 2011/068803 andRhizomucor pusillus with an Aspergillus niger glucoamylase linker andstarch-binding domain (SBD), disclosed SEQ ID NO: 3 in WO 2013/006756with the following substitutions: G128D+D143N.

Commercially available compositions comprising glucoamylase include AMG200 L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME® PLUS, SPIRIZYME®FUEL, SPIRIZYME® B4U, SPIRIZYME® ULTRA, SPIRIZYME® EXCEL, SPIRIZYMEACHIEVE®, and AMG® E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417(from DuPont-Danisco); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™G900, G-ZYME™ and G990 ZR (from DuPont-Danisco).

In one embodiment, the glucoamylase is derived from the Debaryomycesoccidentalis glucoamylase of SEQ ID NO: 102. In one embodiment, theglucoamylase is derived from the Saccharomycopsis fibuligeraglucoamylase of SEQ ID NO: 103. In one embodiment, the glucoamylase isderived from the Saccharomycopsis fibuligera glucoamylase of SEQ ID NO:104. In one embodiment, the glucoamylase is derived from theSaccharomyces cerevisiae glucoamylase of SEQ ID NO: 105. In oneembodiment, the glucoamylase is derived from the Aspergillus nigerglucoamylase of SEQ ID NO: 106. In one embodiment, the glucoamylase isderived from the Aspergillus oryzae glucoamylase of SEQ ID NO: 107. Inone embodiment, the glucoamylase is derived from the Rhizopus oryzaeglucoamylase of SEQ ID NO: 108. In one embodiment, the glucoamylase isderived from the Clostridium thermocellum glucoamylase of SEQ ID NO:109. In one embodiment, the glucoamylase is derived from the Clostridiumthermocellum glucoamylase of SEQ ID NO: 110. In one embodiment, theglucoamylase is derived from the Arxula adeninivorans glucoamylase ofSEQ ID NO: 111. In one embodiment, the glucoamylase is derived from theHormoconis resinae glucoamylase of SEQ ID NO: 112. In one embodiment,the glucoamylase is derived from the Aureobasidium pullulansglucoamylase of SEQ ID NO: 113.

In one embodiment, the glucoamylase is a Trichoderma reeseiglucoamylase, such as the Trichoderma reesei glucoamylase of SEQ ID NO:230.

In one embodiment, the glucoamylase has a Relative Activity heatstability at 85° C. of at least 20%, at least 30%, or at least 35%determined as described in Example 4 of WO2018/098381 (heat stability).

In one embodiment, the glucoamylase has a relative activity pH optimumat pH 5.0 of at least 90%, e.g., at least 95%, at least 97%, or 100%determined as described in Example 4 of WO2018/098381 (pH optimum).

In one embodiment, the glucoamylase has a pH stability at pH 5.0 of atleast 80%, at least 85%, at least 90% determined as described in Example4 of WO2018/098381 (pH stability).

In one embodiment, the glucoamylase used in liquefaction, such as aPenicillium oxalicum glucoamylase variant, has a thermostabilitydetermined as DSC Td at pH 4.0 as described in Example 15 ofWO2018/098381 of at least 70° C., preferably at least 75° C., such as atleast 80° C., such as at least 81° C., such as at least 82° C., such asat least 83° C., such as at least 84° C., such as at least 85° C., suchas at least 86° C., such as at least 87%, such as at least 88° C., suchas at least 89° C., such as at least 90° C. In one embodiment, theglucoamylase, such as a Penicillium oxalicum glucoamylase variant, has athermostability determined as DSC Td at pH 4.0 as described in Example15 of WO2018/098381 in the range between 70° C. and 95° C., such asbetween 80° C. and 90° C.

In one embodiment, the glucoamylase, such as a Penicillium oxalicumglucoamylase variant, used in liquefaction has a thermostabilitydetermined as DSC Td at pH 4.8 as described in Example 15 ofWO2018/098381 of at least 70° C., preferably at least 75° C., such as atleast 80° C., such as at least 81° C., such as at least 82° C., such asat least 83° C., such as at least 84° C., such as at least 85° C., suchas at least 86° C., such as at least 87%, such as at least 88° C., suchas at least 89° C., such as at least 90° C., such as at least 91° C. Inone embodiment, the glucoamylase, such as a Penicillium oxalicumglucoamylase variant, has a thermostability determined as DSC Td at pH4.8 as described in Example 15 of WO2018/098381 in the range between 70°C. and 95° C., such as between 80° C. and 90° C.

In one embodiment, the glucoamylase, such as a Penicillium oxalicumglucoamylase variant, used in liquefaction has a residual activitydetermined as described in Example 16 of WO2018/098381, of at least 100%such as at least 105%, such as at least 110%, such as at least 115%,such as at least 120%, such as at least 125%. In one embodiment, theglucoamylase, such as a Penicillium oxalicum glucoamylase variant, has athermostability determined as residual activity as described in Example16 of WO2018/098381, in the range between 100% and 130%.

In one embodiment, the glucoamylase, e.g., of fungal origin such as afilamentous fungi, from a strain of the genus Penicillium, e.g., astrain of Penicillium oxalicum, in particular the Penicillium oxalicumglucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 (which ishereby incorporated by reference).

In one embodiment, the glucoamylase has a mature polypeptide sequence ofat least 80%, e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identity to the maturepolypeptide shown in SEQ ID NO: 2 in WO 2011/127802.

In one embodiment, the glucoamylase is a variant of the Penicilliumoxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802,having a K79V substitution. The K79V glucoamylase variant has reducedsensitivity to protease degradation relative to the parent as disclosedin WO 2013/036526 (which is hereby incorporated by reference).

In one embodiment, the glucoamylase is derived from Penicilliumoxalicum.

In one embodiment, the glucoamylase is a variant of the Penicilliumoxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802. Inone embodiment, the Penicillium oxalicum glucoamylase is the onedisclosed as SEQ ID NO: 2 in WO 2011/127802 having Val (V) in position79.

Contemplated Penicillium oxalicum glucoamylase variants are disclosed inWO 2013/053801 which is hereby incorporated by reference.

In one embodiment, these variants have reduced sensitivity to proteasedegradation.

In one embodiment, these variant have improved thermostability comparedto the parent.

In one embodiment, the glucoamylase has a K79V substitution (using SEQID NO: 2 of WO 2011/127802 for numbering), corresponding to the PE001variant, and further comprises one of the following alterations orcombinations of alterations

T65A; Q327F; E501V; Y504T; Y504*; T65A+Q327F; T65A+E501V; T65A+Y504T;T65A+Y504*; Q327F+E501V; Q327F+Y504T; Q327F+Y504*; E501V+Y504T;E501V+Y504*; T65A+Q327F+E501V; T65A+Q327F+Y504T; T65A+E501V+Y504T;Q327F+E501V+Y504T; T65A+Q327F+Y504*; T65A+E501V+Y504*;Q327F+E501V+Y504*; T65A+Q327F+E501V+Y504T; T65A+Q327F+E501V+Y504*;E501V+Y504T; T65A+K161S; T65A+Q405T; T65A+Q327W; T65A+Q327F; T65A+Q327Y;P11F+T65A+Q327F; R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F;P2N+P4S+P11F+T65A+Q327F; P11F+D26C+K33C+T65A+Q327F;P2N+P4S+P11F+T65A+Q327W+E501V+Y504T;R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; P11F+T65A+Q327W;P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; P11F+T65A+Q327W+E501V+Y504T;T65A+Q327F+E501V+Y504T; T65A+S105P+Q327W; T65A+S105P+Q327F;T65A+Q327W+S364P; T65A+Q327F+S364P; T65A+S103N+Q327F;P2N+P4S+P11F+K34Y+T65A+Q327F; P2N+P4S+P11F+T65A+Q327F+D445N+V447S;P2N+P4S+P11F+T65A+I172V+Q327F; P2N+P4S+P11F+T65A+Q327F+N502*;P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E;P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S;P2N+P4S+P11F+T65A+Q327F+S377T; P2N+P4S+P11F+T65A+V325T+Q327W;P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T;P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T;P2N+P4S+P11F+D26N+K34Y+T65A+Q327F;P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T;P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;P2N+P4S+T10D+T65A+Q327F+E501V+Y504T;P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; K5A+P11F+T65A+Q327F+E501V+Y504T;P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T;P2N+T10E+E18N+T65A+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N;P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A;P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T;P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T;P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*;P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;K5A+P11F+T65A+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+V791+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; S255N+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T;P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; andP2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

In one embodiment, the Penicillium oxalicum glucoamylase variant has aK79V substitution (using SEQ ID NO: 2 of WO 2011/127802 for numbering),corresponding to the PE001 variant, and further comprises one of thefollowing substitutions or combinations of substitutions:

P11F+T65A+Q327F;

P2N+P4S+P11F+T65A+Q327F;

P11F+D26C+K33C+T65A+Q327F;

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T;

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; and

P11F+T65A+Q327W+E501V+Y504T.

Additional glucoamylases contemplated for use with the present inventioncan be found in WO2011/153516 (the content of which is incorporatedherein).

Additional polynucleotides encoding suitable glucoamylases may beobtained from microorganisms of any genus, including those readilyavailable within the UniProtKB database (www.uniprot.org).

The glucoamylase coding sequences can also be used to design nucleicacid probes to identify and clone DNA encoding glucoamylases fromstrains of different genera or species, as described supra.

The polynucleotides encoding glucoamylases may also be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encodingglucoamylases are described supra.

In one embodiment, the glucoamylase has a mature polypeptide sequencethat comprises or consists of the amino acid sequence of any one of theglucoamylases described or referenced herein (e.g., any one of SEQ IDNOs: 8, 102-113, 229 and 230). In another embodiment, the glucoamylasehas a mature polypeptide sequence that is a fragment of the any one ofthe glucoamylases described or referenced herein (e.g., any one of SEQID NOs: 8, 102-113, 229 and 230). In one embodiment, the number of aminoacid residues in the fragment is at least 75%, e.g., at least 80%, 85%,90%, or 95% of the number of amino acid residues in referenced fulllength glucoamylase (e.g. any one of SEQ ID NOs: 8, 102-113, 229 and230). In other embodiments, the glucoamylase may comprise the catalyticdomain of any glucoamylase described or referenced herein (e.g., thecatalytic domain of any one of SEQ ID NOs: 8, 102-113, 229 and 230).

The glucoamylase may be a variant of any one of the glucoamylasesdescribed supra (e.g., any one of SEQ ID NOs: 8, 102-113, 229 and 230).In one embodiment, the glucoamylase has a mature polypeptide sequence ofat least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, 99%, or 100% sequence identity to any one of the glucoamylasesdescribed supra (e.g., any one of SEQ ID NOs: 8, 102-113, 229 and 230).

In one embodiment, the glucoamylase has a mature polypeptide sequencethat differs by no more than ten amino acids, e.g., by no more than fiveamino acids, by no more than four amino acids, by no more than threeamino acids, by no more than two amino acids, or by one amino acid fromthe amino acid sequence of any one of the glucoamylases described supra(e.g., any one of SEQ ID NOs: 8, 102-113, 229 and 230). In oneembodiment, the glucoamylase has an amino acid substitution, deletion,and/or insertion of one or more (e.g., two, several) of amino acidsequence of any one of the glucoamylases described supra (e.g., any oneof SEQ ID NOs: 8, 102-113, 229 and 230). In some embodiments, the totalnumber of amino acid substitutions, deletions and/or insertions is notmore than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1.

In some embodiments, the glucoamylase has at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the glucoamylase activity of any glucoamylase describedor referenced herein (e.g., any one of SEQ ID NOs: 8, 102-113, 229 and230) under the same conditions.

In one embodiment, the glucoamylase coding sequence hybridizes under atleast low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any glucoamylase described or referenced herein(e.g., any one of SEQ ID NOs: 8, 102-113, 229 and 230). In oneembodiment, the glucoamylase coding sequence has at least 65%, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the coding sequence from any glucoamylasedescribed or referenced herein (e.g., any one of SEQ ID NOs: 8, 102-113,229 and 230).

In one embodiment, the glucoamylase comprises the coding sequence of anyglucoamylase described or referenced herein (any one of SEQ ID NOs: 8,102-113, 229 and 230). In one embodiment, the glucoamylase comprises acoding sequence that is a subsequence of the coding sequence from anyglucoamylase described or referenced herein, wherein the subsequenceencodes a polypeptide having glucoamylase activity. In one embodiment,the number of nucleotides residues in the subsequence is at least 75%,e.g., at least 80%, 85%, 90%, or 95% of the number of the referencedcoding sequence.

The referenced coding sequence of any related aspect or embodimentdescribed herein can be the native coding sequence or a degeneratesequence, such as a codon-optimized coding sequence designed for use ina particular host cell (e.g., optimized for expression in Saccharomycescerevisiae).

The glucoamylase can also include fused polypeptides or cleavable fusionpolypeptides, as described supra.

Proteases

The expressed and/or exogenous protease can be any protease that issuitable for the fermenting organisms and/or their methods of usedescribed herein, such as a naturally occurring protease or a variantthereof that retains protease activity. Any protease contemplated forexpression by a fermenting organism described below is also contemplatedfor aspects of the invention involving exogenous addition of a protease.

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

Protease activity can be measured using any suitable assay, in which asubstrate is employed, that includes peptide bonds relevant for thespecificity of the protease in question. Assay-pH and assay-temperatureare likewise to be adapted to the protease in question. Examples ofassay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples ofassay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80° C.

In some aspects, the fermenting organism comprising a heterologouspolynucleotide encoding a protease has an increased level of proteaseactivity compared to the fermenting organism without the heterologouspolynucleotide encoding the protease, when cultivated under the sameconditions. In some aspects, the fermenting organism has an increasedlevel of protease activity of at least 5%, e.g., at least 10%, at least15%, at least 20%, at least 25%, at least 50%, at least 100%, at least150%, at least 200%, at least 300%, or at 500% compared to thefermenting organism without the heterologous polynucleotide encoding theprotease, when cultivated under the same conditions.

Exemplary proteases that may be expressed with the fermenting organismsand used with the methods described herein include, but are not limitedto, proteases shown in Table 4 (or derivatives thereof).

TABLE 4 Donor Organism SEQ ID NO: (catalytic domain) (maturepolypeptide) Family Aspergillus niger 9 A1 Trichoderma reesei 10Thermoascus aurantiacus 11 M35 Dichomitus squalens 12 S53 Nocardiopsisprasina 13 S1 Penicillium simplicissimum 14 S10 Aspergillus niger 15Meriphilus giganteus 16 S53 Lecanicillium sp. WMM742 17 S53 Talaromycesproteolyticus 18 S53 Penicillium ranomafanaense 19 A1A Aspergillusoryzae 20 S53 Talaromyces liani 21 S10 Thermoascus thermophilus 22 S53Pyrococcus furiosus 23 Trichoderma reesei 24 Rhizomucor miehei 25Lenzites betulinus 26 S53 Neolentinus lepideus 27 S53 Thermococcus sp.28 S8 Thermococcus sp. 29 S8 Thermomyces lanuginosus 30 S53 Thermococcusthioreducens 31 S53 Polyporus arcularius 32 S53 Ganoderma lucidum 33 S53Ganoderma lucidum 34 S53 Ganoderma lucidum 35 S53 Trametes sp. AH28-2 36S53 Cinereomyces lindbladii 37 S53 Trametes versicolor O82DDP 38 S53Paecilomyces hepiali 39 S53 Isaria tenuipes 40 S53 Aspergillus tamarii41 S53 Aspergillus brasiliensis 42 S53 Aspergillus iizukae 43 S53Penicillium sp-72364 44 S10 Aspergillus denticulatus 45 S10 Hamigera sp.t184-6 46 S10 Penicillium janthinellum 47 S10 Penicillium vasconiae 48S10 Hamigera paravellanea 49 S10 Talaromyces variabilis 50 S10Penicillium arenicola 51 S10 Nocardiopsis kunsanensis 52 S1 Streptomycesparvulus 53 S1 Saccharopolyspora endophytica 54 S1 luteus cellwallenrichments K 55 S1 Saccharothrix australiensis 56 S1 Nocardiopsisbaichengensis 57 S1 Streptomyces sp. SM15 58 S1 Actinoalloteichusspitiensis 59 S1 Byssochlamys verrucosa 60 M35 Hamigera terricola 61 M35Aspergillus tamarii 62 M35 Aspergillus niveus 63 M35 Penicilliumsclerotiorum 64 A1 Penicillium bilaiae 65 A1 Penicillium antarcticum 66A1 Penicillium sumatrense 67 A1 Trichoderma lixii 68 A1 Trichodermabrevicompactum 69 A1 Penicillium cinnamopurpureum 70 A1 Bacilluslicheniformis 71 S8 Bacillus subtilis 72 S8 Trametes cf versicol 73 S53

Additional polynucleotides encoding suitable proteases may be derivedfrom microorganisms of any suitable genus, including those readilyavailable within the UniProtKB database (www.uniprot.org).

In one embodiment, the protease is derived from Aspergillus, such as theAspergillus niger protease of SEQ ID NO: 9, the Aspergillus tamariiprotease of SEQ ID NO: 41, or the Aspergillus denticulatus protease ofSEQ ID NO: 45. In one embodiment, the protease is derived fromDichomitus, such as the Dichomitus squalens protease of SEQ ID NO: 12.In one embodiment, the protease is derived from Penicillium, such as thePenicillium simplicissimum protease of SEQ ID NO: 14, the Penicilliumantarcticum protease of SEQ ID NO: 66, or the Penicillium sumatrenseprotease of SEQ ID NO: 67. In one aspect, the protease is derived fromMeriphilus, such as the Meriphilus giganteus protease of SEQ ID NO: 16.In one aspect, the protease is derived from Talaromyces, such as theTalaromyces liani protease of SEQ ID NO: 21. In one aspect, the proteaseis derived from Thermoascus, such as the Thermoascus thermophilusprotease of SEQ ID NO: 22. In one aspect, the protease is derived fromGanoderma, such as the Ganoderma lucidum protease of SEQ ID NO: 33. Inone aspect, the protease is derived from Hamigera, such as the Hamigeraterricola protease of SEQ ID NO: 61. In one aspect, the protease isderived from Trichoderma, such as the Trichoderma brevicompactumprotease of SEQ ID NO: 69.

The protease coding sequences can also be used to design nucleic acidprobes to identify and clone DNA encoding proteases from strains ofdifferent genera or species, as described supra.

The polynucleotides encoding proteases may also be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encoding proteasesare described supra.

In one embodiment, the protease has a mature polypeptide sequence thatcomprises or consists of the amino acid sequence of any one of SEQ IDNOs: 9-73 (e.g., any one of SEQ ID NOs: 9, 14, 16, 21, 22, 33, 41, 45,61, 62, 66, 67, and 69; such as any one of SEQ NOs: 9, 14, 16, and 69).In another embodiment, the protease has a mature polypeptide sequencethat is a fragment of the protease of any one of SEQ ID NOs: 9-73 (e.g.,wherein the fragment has protease activity). In one embodiment, thenumber of amino acid residues in the fragment is at least 75%, e.g., atleast 80%, 85%, 90%, or 95% of the number of amino acid residues inreferenced full length protease (e.g. any one of SEQ ID NOs: 9-73). Inother embodiments, the protease may comprise the catalytic domain of anyprotease described or referenced herein (e.g., the catalytic domain ofany one of SEQ ID NOs: 9-73).

The protease may be a variant of any one of the proteases describedsupra (e.g., any one of SEQ ID NOs: 9-73. In one embodiment, theprotease has a mature polypeptide sequence of at least 60%, e.g., atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity to any one of the proteases described supra (e.g., any one ofSEQ ID NOs: 9-73).

In one embodiment, the protease has a mature polypeptide sequence thatdiffers by no more than ten amino acids, e.g., by no more than fiveamino acids, by no more than four amino acids, by no more than threeamino acids, by no more than two amino acids, or by one amino acid fromthe amino acid sequence of any one of the proteases described supra(e.g., any one of SEQ ID NOs: 9-73). In one embodiment, the protease hasan amino acid substitution, deletion, and/or insertion of one or more(e.g., two, several) of amino acid sequence of any one of the proteasesdescribed supra (e.g., any one of SEQ ID NOs: 9-73). In someembodiments, the total number of amino acid substitutions, deletionsand/or insertions is not more than 10, e.g., not more than 9, 8, 7, 6,5, 4, 3, 2, or 1.

In one embodiment, the protease coding sequence hybridizes under atleast low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any protease described or referenced herein(e.g., any one of SEQ ID NOs: 9-73). In one embodiment, the proteasecoding sequence has at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe coding sequence from any protease described or referenced herein(e.g., any one of SEQ ID NOs: 9-73).

In one embodiment, the protease comprises the coding sequence of anyprotease described or referenced herein (any one of SEQ ID NOs: 9-73).In one embodiment, the protease comprises a coding sequence that is asubsequence of the coding sequence from any protease described orreferenced herein, wherein the subsequence encodes a polypeptide havingprotease activity. In one embodiment, the number of nucleotides residuesin the subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95%of the number of the referenced coding sequence.

The referenced coding sequence of any related aspect or embodimentdescribed herein can be the native coding sequence or a degeneratesequence, such as a codon-optimized coding sequence designed for use ina particular host cell (e.g., optimized for expression in Saccharomycescerevisiae).

The protease can also include fused polypeptides or cleavable fusionpolypeptides, as described supra.

In one embodiment, the protease used according to a process describedherein is a Serine proteases. In one particular embodiment, the proteaseis a serine protease belonging to the family 53, e.g., an endo-protease,such as S53 protease from Meripilus giganteus, Dichomitus squalensTrametes versicolor, Polyporus arcularius, Lenzites betulinus, Ganodermalucidum, Neolentinus lepideus, or Bacillus sp. 19138, in a process forproducing ethanol from a starch-containing material, the ethanol yieldwas improved, when the S53 protease was present/or added duringsaccharification and/or fermentation of either gelatinized orun-gelatinized starch. In one embodiment, the proteases is selectedfrom: (a) proteases belonging to the EC 3.4.21 enzyme group; and/or (b)proteases belonging to the EC 3.4.14 enzyme group; and/or (c) Serineproteases of the peptidase family S53 that comprises two different typesof peptidases: tripeptidyl aminopeptidases (exo-type) andendo-peptidases; as described in 1993, Biochem. J. 290:205-218 and inMEROPS protease database, release, 9.4 (31 Jan. 2011)(www.merops.ac.uk). The database is described in Rawlings, N. D.,Barrett, A. J. and Bateman, A., 2010, “MEROPS: the peptidase database”,Nucl. Acids Res. 38: D227-D233.

For determining whether a given protease is a Serine protease, and afamily S53 protease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Peptidase family S53 contains acid-acting endopeptidases andtripeptidyl-peptidases. The residues of the catalytic triad are Glu,Asp, Ser, and there is an additional acidic residue, Asp, in theoxyanion hole. The order of the residues is Glu, Asp, Asp, Ser. The Serresidue is the nucleophile equivalent to Ser in the Asp, His, Ser triadof subtilisin, and the Glu of the triad is a substitute for the generalbase, His, in subtilisin.

The peptidases of the S53 family tend to be most active at acidic pH(unlike the homologous subtilisins), and this can be attributed to thefunctional importance of carboxylic residues, notably Asp in theoxyanion hole. The amino acid sequences are not closely similar to thosein family S8 (i.e. serine endopeptidase subtilisins and homologues), andthis, taken together with the quite different active site residues andthe resulting lower pH for maximal activity, provides for a substantialdifference to that family. Protein folding of the peptidase unit formembers of this family resembles that of subtilisin, having the clantype SB.

In one embodiment, the protease used according to a process describedherein is a Cysteine proteases.

In one embodiment, the protease used according to a process describedherein is a Aspartic proteases. Aspartic acid proteases are describedin, for example, Hand-book of Proteolytic En-zymes, Edited by A. J.Barrett, N. D. Rawlings and J. F. Woessner, Aca-demic Press, San Diego,1998, Chapter 270). Suitable examples of aspartic acid protease include,e.g., those disclosed in R. M. Berka et al. Gene, 96, 313 (1990)); (R.M. Berka et al. Gene, 125, 195-198 (1993)); and Gomi et al. Biosci.Biotech. Biochem. 57, 1095-1100 (1993), which are hereby incorporated byreference.

The protease also may be a metalloprotease, which is defined as aprotease selected from the group consisting of:

(a) proteases belonging to EC 3.4.24 (metalloendopeptidases); preferablyEC 3.4.24.39 (acid metallo proteinases);

(b) metalloproteases belonging to the M group of the above Handbook;

(c) metalloproteases not yet assigned to clans (designation: Clan MX),or belonging to either one of clans MA, MB, MC, MD, ME, MF, MG, MH (asdefined at pp. 989-991 of the above Handbook);

(d) other families of metalloproteases (as defined at pp. 1448-1452 ofthe above Handbook);

(e) metalloproteases with a HEXXH motif;

(f) metalloproteases with an HEFTH motif;

(g) metalloproteases belonging to either one of families M3, M26, M27,M32, M34, M35, M36, M41, M43, or M47 (as defined at pp. 1448-1452 of theabove Handbook);

(h) metalloproteases belonging to the M28E family; and

(i) metalloproteases belonging to family M35 (as defined at pp.1492-1495 of the above Handbook).

In other particular embodiments, metalloproteases are hydrolases inwhich the nucleophilic attack on a peptide bond is mediated by a watermolecule, which is activated by a divalent metal cation. Examples ofdivalent cations are zinc, cobalt or manganese. The metal ion may beheld in place by amino acid ligands. The number of ligands may be five,four, three, two, one or zero. In a particular embodiment the number istwo or three, preferably three.

There are no limitations on the origin of the metalloprotease used in aprocess of the invention. In an embodiment the metalloprotease isclassified as EC 3.4.24, preferably EC 3.4.24.39. In one embodiment, themetalloprotease is an acid-stable metalloprotease, e.g., a fungalacid-stable metalloprotease, such as a metalloprotease derived from astrain of the genus Thermoascus, preferably a strain of Thermoascusaurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670(classified as EC 3.4.24.39). In another embodiment, the metalloproteaseis derived from a strain of the genus Aspergillus, preferably a strainof Aspergillus oryzae.

In one embodiment the metalloprotease has a degree of sequence identityto amino acids −178 to 177, −159 to 177, or preferably amino acids 1 to177 (the mature polypeptide) of SEQ ID NO: 1 of WO 2010/008841 (aThermoascus aurantiacus metalloprotease) of at least 80%, at least 82%,at least 85%, at least 90%, at least 95%, or at least 97%; and whichhave metalloprotease activity. In particular embodiments, themetalloprotease consists of an amino acid sequence with a degree ofidentity to SEQ ID NO: 1 as mentioned above.

The Thermoascus aurantiacus metalloprotease is a preferred example of ametalloprotease suitable for use in a process of the invention. Anothermetalloprotease is derived from Aspergillus oryzae and comprises thesequence of SEQ ID NO: 11 disclosed in WO 2003/048353, or amino acids−23-353; −23-374; −23-397; 1-353; 1-374; 1-397; 177-353; 177-374; or177-397 thereof, and SEQ ID NO: 10 disclosed in WO 2003/048353.

Another metalloprotease suitable for use in a process of the inventionis the Aspergillus oryzae metalloprotease comprising SEQ ID NO: 5 of WO2010/008841, or a metalloprotease is an isolated polypeptide which has adegree of identity to SEQ ID NO: 5 of at least about 80%, at least 82%,at least 85%, at least 90%, at least 95%, or at least 97%; and whichhave metalloprotease activity. In particular embodiments, themetalloprotease consists of the amino acid sequence of SEQ ID NO: 5 ofWO 2010/008841.

In a particular embodiment, a metalloprotease has an amino acid sequencethat differs by forty, thirty-five, thirty, twenty-five, twenty, or byfifteen amino acids from amino acids −178 to 177, −159 to 177, or +1 to177 of the amino acid sequences of the Thermoascus aurantiacus orAspergillus oryzae metalloprotease.

In another embodiment, a metalloprotease has an amino acid sequence thatdiffers by ten, or by nine, or by eight, or by seven, or by six, or byfive amino acids from amino acids −178 to 177, −159 to 177, or +1 to 177of the amino acid sequences of these metalloproteases, e.g., by four, bythree, by two, or by one amino acid.

In particular embodiments, the metalloprotease a) comprises or b)consists of

i) the amino acid sequence of amino acids −178 to 177, −159 to 177, or+1 to 177 of SEQ ID NO:1 of WO 2010/008841;

ii) the amino acid sequence of amino acids −23-353, −23-374, −23-397,1-353, 1-374, 1-397, 177-353, 177-374, or 177-397 of SEQ ID NO: 3 of WO2010/008841;

iii) the amino acid sequence of SEQ ID NO: 5 of WO 2010/008841; orallelic variants, or fragments, of the sequences of i), ii), and iii)that have protease activity.

A fragment of amino acids −178 to 177, −159 to 177, or +1 to 177 of SEQID NO: 1 of WO 2010/008841 or of amino acids −23-353, −23-374, −23-397,1-353, 1-374, 1-397, 177-353, 177-374, or 177-397 of SEQ ID NO: 3 of WO2010/008841; is a polypeptide having one or more amino acids deletedfrom the amino and/or carboxyl terminus of these amino acid sequences.In one embodiment a fragment contains at least 75 amino acid residues,or at least 100 amino acid residues, or at least 125 amino acidresidues, or at least 150 amino acid residues, or at least 160 aminoacid residues, or at least 165 amino acid residues, or at least 170amino acid residues, or at least 175 amino acid residues.

To determine whether a given protease is a metallo protease or not,reference is made to the above “Handbook of Proteolytic Enzymes” and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

The protease may be a variant of, e.g., a wild-type protease, havingthermostability properties defined herein. In one embodiment, thethermostable protease is a variant of a metallo protease. In oneembodiment, the thermostable protease used in a process described hereinis of fungal origin, such as a fungal metallo protease, such as a fungalmetallo protease derived from a strain of the genus Thermoascus,preferably a strain of Thermoascus aurantiacus, especially Thermoascusaurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39).

In one embodiment, the thermostable protease is a variant of the maturepart of the metallo protease shown in SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 furtherwith one of the following substitutions or combinations ofsubstitutions:

S5*+D79L+S87P+A112P+D142L;

D79L+S87P+A112P+T124V+D142L;

S5*+N26R+D79L+S87P+A112P+D142L;

N26R+T46R+D79L+S87P+A112P+D142L;

T46R+D79L+S87P+T116V+D142L;

D79L+P81R+S87P+A112P+D142L;

A27K+D79L+S87P+A112P+T124V+D142L;

D79L+Y82F+S87P+A112P+T124V+D142L;

D79L+Y82F+S87P+A112P+T124V+D142L;

D79L+S87P+A112P+T124V+A126V+D142L;

D79L+S87P+A112P+D142L;

D79L+Y82F+S87P+A112P+D142L;

S38T+D79L+S87P+A112P+A126V+D142L;

D79L+Y82F+S87P+A112P+A126V+D142L;

A27K+D79L+S87P+A112P+A126V+D142L;

D79L+S87P+N98C+A112P+G135C+D142L;

D79L+S87P+A112P+D142L+T141C+M1610;

S36P+D79L+S87P+A112P+D142L;

A37P+D79L+S87P+A112P+D142L;

S49P+D79L+S87P+A112P+D142L;

S50P+D79L+S87P+A112P+D142L;

D79L+S87P+D104P+A112P+D142L;

D79L+Y82F+S87G+A112P+D142L;

S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;

D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;

S70V+D79L+Y82F+S87G+A112P+D142L;

D79L+Y82F+S87G+D104P+A112P+D142L;

D79L+Y82F+S87G+A112P+A126V+D142L;

Y82F+S87G+S70V+D79L+D104P+A112P+D142L;

Y82F+S87G+D79L+D104P+A112P+A126V+D142L;

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;

A27K+Y82F+S87G+D104P+A112P+A126V+D142L;

A27K+D79L+Y82F+D104P+A112P+A126V+D142L;

A27K+Y82F+D104P+A112P+A126V+D142L;

A27K+D79L+S87P+A112P+D142L; and

D79L+S87P+D142L.

In one embodiment, the thermostable protease is a variant of the metalloprotease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 withone of the following substitutions or combinations of substitutions:

D79L+S87P+A112P+D142L;

D79L+S87P+D142L; and

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In one embodiment, the protease variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841.

The thermostable protease may also be derived from any bacterium as longas the protease has the thermostability properties.

In one embodiment, the thermostable protease is derived from a strain ofthe bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfuprotease).

In one embodiment, the protease is one shown as SEQ ID NO: 1 in U.S.Pat. No. 6,358,726-B1 (Takara Shuzo Company).

In one embodiment, the thermostable protease is a protease having amature polypeptide sequence of at least 80% identity, such as at least85%, such as at least 90%, such as at least 95%, such as at least 96%,such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1. The Pyroccusfuriosus protease can be purchased from Takara Bio, Japan.

The Pyrococcus furiosus protease may be a thermostable protease asdescribed in SEQ ID NO: 13 of WO2018/098381. This protease (PfuS) wasfound to have a thermostability of 110% (80° C./70° C.) and 103% (90°C./70° C.) at pH 4.5 determined.

In one embodiment a thermostable protease used in a process describedherein has a thermostability value of more than 20% determined asRelative Activity at 80° C./70° C. determined as described in Example 2of WO2018/098381.

In one embodiment, the protease has a thermostability of more than 30%,more than 40%, more than 50%, more than 60%, more than 70%, more than80%, more than 90%, more than 100%, such as more than 105%, such as morethan 110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70° C.

In one embodiment, protease has a thermostability of between 20 and 50%,such as between 20 and 40%, such as 20 and 30% determined as RelativeActivity at 80° C./70° C. In one embodiment, the protease has athermostability between 50 and 115%, such as between 50 and 70%, such asbetween 50 and 60%, such as between 100 and 120%, such as between 105and 115% determined as Relative Activity at 80° C./70° C.

In one embodiment, the protease has a thermostability value of more than10% determined as Relative Activity at 85° C./70° C. determined asdescribed in Example 2 of WO2018/098381.

In one embodiment, the protease has a thermostability of more than 10%,such as more than 12%, more than 14%, more than 16%, more than 18%, morethan 20%, more than 30%, more than 40%, more that 50%, more than 60%,more than 70%, more than 80%, more than 90%, more than 100%, more than110% determined as Relative Activity at 85° C./70° C.

In one embodiment, the protease has a thermostability of between 10% and50%, such as between 10% and 30%, such as between 10% and 25% determinedas Relative Activity at 85° C./70° C.

In one embodiment, the protease has more than 20%, more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% determined as Remaining Activity at 80° C.; and/or theprotease has more than 20%, more than 30%, more than 40%, more than 50%,more than 60%, more than 70%, more than 80%, more than 90% determined asRemaining Activity at 84° C.

Determination of “Relative Activity” and “Remaining Activity” is done asdescribed in Example 2 of WO2018/098381.

In one embodiment, the protease may have a themostability for above 90,such as above 100 at 85° C. as determined using the Zein-BCA assay asdisclosed in Example 3 of WO2018/098381.

In one embodiment, the protease has a themostability above 60%, such asabove 90%, such as above 100%, such as above 110% at 85° C. asdetermined using the Zein-BCA assay of WO2018/098381.

In one embodiment, protease has a themostability between 60-120, such asbetween 70-120%, such as between 80-120%, such as between 90-120%, suchas between 100-120%, such as 110-120% at 85° C. as determined using theZein-BCA assay of WO2018/098381.

In one embodiment, the thermostable protease has at least 20%, such asat least 30%, such as at least 40%, such as at least 50%, such as atleast 60%, such as at least 70%, such as at least 80%, such as at least90%, such as at least 95%, such as at least 100% of the activity of theJTP196 protease variant or Protease Pfu determined by the AZCL-caseinassay of WO2018/098381, and described herein.

In one embodiment, the thermostable protease has at least 20%, such asat least 30%, such as at least 40%, such as at least 50%, such as atleast 60%, such as at least 70%, such as at least 80%, such as at least90%, such as at least 95%, such as at least 100% of the proteaseactivity of the Protease 196 variant or Protease Pfu determined by theAZCL-casein assay of WO2018/098381, and described herein.

Pullulanases

In some embodiments, a pullulanase is present and/or added inliquefaction step and/or saccharification step, or simultaneoussaccharification and fermentation (SSF).

Pullulanases (E.C. 3.2.1.41, pullulan 6-glucano-hydrolase), aredebranching enzymes characterized by their ability to hydrolyze thealpha-1,6-glycosidic bonds in, for example, amylopectin and pullulan.

In some embodiments, the fermenting organism comprises a heterologouspolynucleotide encoding a pullulanase. Any pullulanase described orreferenced herein is contemplated for expression in the fermentingorganism.

The pullulanase may be any pullulanase that is suitable for the hostcells and/or the methods described herein, such as a naturally occurringpullulanase or a variant thereof that retains pullulanase activity.

In some embodiments, the fermenting organism comprising a heterologouspolynucleotide encoding a pullulanase has an increased level ofpullulanase activity compared to the host cells without the heterologouspolynucleotide encoding the pullulanase, when cultivated under the sameconditions. In some embodiments, the fermenting organism has anincreased level of pullulanase activity of at least 5%, e.g., at least10%, at least 15%, at least 20%, at least 25%, at least 50%, at least100%, at least 150%, at least 200%, at least 300%, or at 500% comparedto the fermenting organism without the heterologous polynucleotideencoding the pullulanase, when cultivated under the same conditions.

Exemplary pullulanasees that can be used with the host cells and/or themethods described herein include bacterial, yeast, or filamentous fungalpullulanases, e.g., obtained from any of the microorganisms described orreferenced herein, as described supra under the sections related toalpha-amylases.

Contemplated pullulanases include the pullulanases from Bacillusamyloderamificans disclosed in U.S. Pat. No. 4,560,651 (herebyincorporated by reference), the pullulanase disclosed as SEQ ID NO: 2 inWO 01/151620 (hereby incorporated by reference), the Bacillusderamificans disclosed as SEQ ID NO: 4 in WO 01/151620 (herebyincorporated by reference), and the pullulanase from Bacillusacidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (herebyincorporated by reference) and also described in FEMS Mic. Let. (1994)115, 97-106.

Additional pullulanases contemplated include the pullulanases fromPyrococcus woesei, specifically from Pyrococcus woesei DSM No. 3773disclosed in WO92/02614.

In one embodiment, the pullulanase is a family GH57 pullulanase. In oneembodiment, the pullulanase includes an X47 domain as disclosed in U.S.61/289,040 published as WO 2011/087836 (which are hereby incorporated byreference). More specifically the pullulanase may be derived from astrain of the genus Thermococcus, including Thermococcus litoralis andThermococcus hydrothermalis, such as the Thermococcus hydrothermalispullulanase truncated at site X4 right after the X47 domain (i.e., aminoacids 1-782). The pullulanase may also be a hybrid of the Thermococcuslitoralis and Thermococcus hydrothermalis pullulanases or a T.hydrothermalis/T. litoralis hybrid enzyme with truncation site X4disclosed in U.S. 61/289,040 published as WO 2011/087836 (which ishereby incorporated by reference).

In another embodiment, the pullulanase is one comprising an X46 domaindisclosed in WO 2011/076123 (Novozymes).

The pullulanase may be added in an effective amount which include thepreferred amount of about 0.0001-10 mg enzyme protein per gram DS,preferably 0.0001-0.10 mg enzyme protein per gram DS, more preferably0.0001-0.010 mg enzyme protein per gram DS. Pullulanase activity may bedetermined as NPUN. An Assay for determination of NPUN is described inWO2018/098381.

Suitable commercially available pullulanase products include PROMOZYMED, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300(DuPont-Danisco, USA), and AMANO 8 (Amano, Japan).

In one embodiment, the pullulanase is derived from the Bacillus subtilispullulanase of SEQ ID NO: 114. In one embodiment, the pullulanase isderived from the Bacillus licheniformis pullulanase of SEQ ID NO: 115.In one embodiment, the pullulanase is derived from the Oryza sativapullulanase of SEQ ID NO: 116. In one embodiment, the pullulanase isderived from the Triticum aestivum pullulanase of SEQ ID NO: 117. In oneembodiment, the pullulanase is derived from the Clostridiumphytofermentans pullulanase of SEQ ID NO: 118. In one embodiment, thepullulanase is derived from the Streptomyces avermitilis pullulanase ofSEQ ID NO: 119. In one embodiment, the pullulanase is derived from theKlebsiella pneumoniae pullulanase of SEQ ID NO: 120.

Additional pullulanases contemplated for use with the present inventioncan be found in WO2011/153516 (the content of which is incorporatedherein).

Additional polynucleotides encoding suitable pullulanases may beobtained from microorganisms of any genus, including those readilyavailable within the UniProtKB database (www.uniprot.org).

The pullulanase coding sequences can also be used to design nucleic acidprobes to identify and clone DNA encoding pullulanases from strains ofdifferent genera or species, as described supra.

The polynucleotides encoding pullulanases may also be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encodingpullulanases are described supra.

In one embodiment, the pullulanase has a mature polypeptide sequencethat comprises or consists of the amino acid sequence of any one of thepullulanases described or referenced herein (e.g., any one of SEQ IDNOs: 114-120). In another embodiment, the pullulanase has a maturepolypeptide sequence that is a fragment of the any one of thepullulanases described or referenced herein (e.g., any one of SEQ IDNOs: 114-120). In one embodiment, the number of amino acid residues inthe fragment is at least 75%, e.g., at least 80%, 85%, 90%, or 95% ofthe number of amino acid residues in referenced full length pullulanase.In other embodiments, the pullulanase may comprise the catalytic domainof any pullulanase described or referenced herein (e.g., any one of SEQID NOs: 114-120).

The pullulanase may be a variant of any one of the pullulanasesdescribed supra (e.g., any one of SEQ ID NOs: 114-120). In oneembodiment, the pullulanase has a mature polypeptide sequence of atleast 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, or 100% sequence identity to any one of the pullulanases describedsupra (e.g., any one of SEQ ID NOs: 114-120).

In one embodiment, the pullulanase has a mature polypeptide sequencethat differs by no more than ten amino acids, e.g., by no more than fiveamino acids, by no more than four amino acids, by no more than threeamino acids, by no more than two amino acids, or by one amino acid fromthe amino acid sequence of any one of the pullulanases described supra(e.g., any one of SEQ ID NOs: 114-120). In one embodiment, thepullulanase has an amino acid substitution, deletion, and/or insertionof one or more (e.g., two, several) of amino acid sequence of any one ofthe pullulanases described supra (e.g., any one of SEQ ID NOs: 114-120).In some embodiments, the total number of amino acid substitutions,deletions and/or insertions is not more than 10, e.g., not more than 9,8, 7, 6, 5, 4, 3, 2, or 1.

In some embodiments, the pullulanase has at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the pullulanase activity of any pullulanase described orreferenced herein under the same conditions (e.g., any one of SEQ IDNOs: 114-120).

In one embodiment, the pullulanase coding sequence hybridizes under atleast low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any pullulanase described or referenced herein(e.g., any one of SEQ ID NOs: 114-120). In one embodiment, thepullulanase coding sequence has at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with the coding sequence from any pullulanase described orreferenced herein (e.g., any one of SEQ ID NOs: 114-120).

In one embodiment, the pullulanase comprises the coding sequence of anypullulanase described or referenced herein (e.g., any one of SEQ ID NOs:114-120). In one embodiment, the pullulanase comprises a coding sequencethat is a subsequence of the coding sequence from any pullulanasedescribed or referenced herein, wherein the subsequence encodes apolypeptide having pullulanase activity. In one embodiment, the numberof nucleotides residues in the subsequence is at least 75%, e.g., atleast 80%, 85%, 90%, or 95% of the number of the referenced codingsequence.

The referenced coding sequence of any related aspect or embodimentdescribed herein can be the native coding sequence or a degeneratesequence, such as a codon-optimized coding sequence designed for use ina particular host cell (e.g., optimized for expression in Saccharomycescerevisiae).

The pullulanase can also include fused polypeptides or cleavable fusionpolypeptides, as described supra.

Methods Using a Cellulosic-Containing Material

In some aspects, the methods described herein produce a fermentationproduct from a cellulosic-containing material. The predominantpolysaccharide in the primary cell wall of biomass is cellulose, thesecond most abundant is hemicellulose, and the third is pectin. Thesecondary cell wall, produced after the cell has stopped growing, alsocontains polysaccharides and is strengthened by polymeric lignincovalently cross-linked to hemicellulose. Cellulose is a homopolymer ofanhydrocellobiose and thus a linear beta-(1-4)-D-glucan, whilehemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic-containing material can be, but is not limited to,agricultural residue, herbaceous material (including energy crops),municipal solid waste, pulp and paper mill residue, waste paper, andwood (including forestry residue) (see, for example, Wiselogel et al.,1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-118,Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25:695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In one embodiment, thecellulosic-containing material is any biomass material. In anotherembodiment, the cellulosic-containing material is lignocellulose, whichcomprises cellulose, hemicelluloses, and lignin.

In one embodiment, the cellulosic-containing material is agriculturalresidue, herbaceous material (including energy crops), municipal solidwaste, pulp and paper mill residue, waste paper, or wood (includingforestry residue).

In another embodiment, the cellulosic-containing material is arundo,bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus, ricestraw, switchgrass, or wheat straw. In another embodiment, thecellulosic-containing material is aspen, eucalyptus, fir, pine, poplar,spruce, or willow.

In another embodiment, the cellulosic-containing material is algalcellulose, bacterial cellulose, cotton linter, filter paper,microcrystalline cellulose (e.g., AVICEL®), or phosphoric-acid treatedcellulose.

In another embodiment, the cellulosic-containing material is an aquaticbiomass. As used herein the term “aquatic biomass” means biomassproduced in an aquatic environment by a photosynthesis process. Theaquatic biomass can be algae, emergent plants, floating-leaf plants, orsubmerged plants.

The cellulosic-containing material may be used as is or may be subjectedto pretreatment, using conventional methods known in the art, asdescribed herein. In a preferred embodiment, the cellulosic-containingmaterial is pretreated.

The methods of using cellulosic-containing material can be accomplishedusing methods conventional in the art. Moreover, the methods of can beimplemented using any conventional biomass processing apparatusconfigured to carry out the processes.

Cellulosic Pretreatment

In one embodiment the cellulosic-containing material is pretreatedbefore saccharification.

In practicing the processes described herein, any pretreatment processknown in the art can be used to disrupt plant cell wall components ofthe cellulosic-containing material (Chandra et al., 2007, Adv. Biochem.Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv. Biochem.Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, BioresourceTechnology 100: 10-18; Mosier et al., 2005, Bioresource Technology 96:673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci. 9: 1621-1651;Yang and Wyman, 2008, Biofuels Bioproducts and Biorefining-Biofpr. 2:26-40).

The cellulosic-containing material can also be subjected to particlesize reduction, sieving, pre-soaking, wetting, washing, and/orconditioning prior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

In a one embodiment, the cellulosic-containing material is pretreatedbefore saccharification (i.e., hydrolysis) and/or fermentation.Pretreatment is preferably performed prior to the hydrolysis.Alternatively, the pretreatment can be carried out simultaneously withenzyme hydrolysis to release fermentable sugars, such as glucose,xylose, and/or cellobiose. In most cases the pretreatment step itselfresults in some conversion of biomass to fermentable sugars (even inabsence of enzymes).

In one embodiment, the cellulosic-containing material is pretreated withsteam. In steam pretreatment, the cellulosic-containing material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic-containingmaterial is passed to or through a reaction vessel where steam isinjected to increase the temperature to the required temperature andpressure and is retained therein for the desired reaction time. Steampretreatment is preferably performed at 140-250° C., e.g., 160-200° C.or 170-190° C., where the optimal temperature range depends on optionaladdition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-60 minutes, e.g., 1-30 minutes, 1-20minutes, 3-12 minutes, or 4-10 minutes, where the optimal residence timedepends on the temperature and optional addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatthe cellulosic-containing material is generally only moist during thepretreatment. The steam pretreatment is often combined with an explosivedischarge of the material after the pretreatment, which is known assteam explosion, that is, rapid flashing to atmospheric pressure andturbulent flow of the material to increase the accessible surface areaby fragmentation (Duff and Murray, 1996, Bioresource Technology 855:1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628;U.S. Patent Application No. 2002/0164730). During steam pretreatment,hemicellulose acetyl groups are cleaved and the resulting acidautocatalyzes partial hydrolysis of the hemicellulose to monosaccharidesand oligosaccharides. Lignin is removed to only a limited extent.

In one embodiment, the cellulosic-containing material is subjected to achemical pretreatment. The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexpansion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic-containingmaterial is mixed with dilute acid, typically H₂SO₄, and water to form aslurry, heated by steam to the desired temperature, and after aresidence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Schell et al., 2004, BioresourceTechnology 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol.65: 93-115). In a specific embodiment the dilute acid pretreatment ofcellulosic-containing material is carried out using 4% w/w sulfuric acidat 180° C. for 5 minutes.

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment. Lime pretreatment isperformed with calcium oxide or calcium hydroxide at temperatures of85-150° C. and residence times from 1 hour to several days (Wyman etal., 2005, Bioresource Technology 96: 1959-1966; Mosier et al., 2005,Bioresource Technology 96: 673-686). WO 2006/110891, WO 2006/110899, WO2006/110900, and WO 2006/110901 disclose pretreatment methods usingammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technology 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating thecellulosic-containing material with liquid or gaseous ammonia atmoderate temperatures such as 90-150° C. and high pressure such as 17-20bar for 5-10 minutes, where the dry matter content can be as high as 60%(Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35;Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh etal., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri et al.,2005, Bioresource Technology 96: 2014-2018). During AFEX pretreatmentcellulose and hemicelluloses remain relatively intact.Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic-containing materialby extraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and U.S. PublishedApplication 2002/0164730.

In one embodiment, the chemical pretreatment is carried out as a diluteacid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acidor 0.1 to 2 wt. % acid. The acid is contacted with thecellulosic-containing material and held at a temperature in the range ofpreferably 140-200° C., e.g., 165-190° C., for periods ranging from 1 to60 minutes.

In another embodiment, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic-containing material is present duringpretreatment in amounts preferably between 10-80 wt. %, e.g., 20-70 wt.% or 30-60 wt. %, such as around 40 wt. %. The pretreatedcellulosic-containing material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

In one embodiment, the cellulosic-containing material is subjected tomechanical or physical pretreatment. The term “mechanical pretreatment”or “physical pretreatment” refers to any pretreatment that promotes sizereduction of particles. For example, such pretreatment can involvevarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

The cellulosic-containing material can be pretreated both physically(mechanically) and chemically. Mechanical or physical pretreatment canbe coupled with steaming/steam explosion, hydrothermolysis, dilute ormild acid treatment, high temperature, high pressure treatment,irradiation (e.g., microwave irradiation), or combinations thereof. Inone aspect, high pressure means pressure in the range of preferablyabout 100 to about 400 psi, e.g., about 150 to about 250 psi. In anotheraspect, high temperature means temperature in the range of about 100 toabout 300° C., e.g., about 140 to about 200° C. In a preferred aspect,mechanical or physical pretreatment is performed in a batch-processusing a steam gun hydrolyzer system that uses high pressure and hightemperature as defined above, e.g., a Sunds Hydrolyzer available fromSunds Defibrator AB, Sweden. The physical and chemical pretreatments canbe carried out sequentially or simultaneously, as desired.

Accordingly, in one embodiment, the cellulosic-containing material issubjected to physical (mechanical) or chemical pretreatment, or anycombination thereof, to promote the separation and/or release ofcellulose, hemicellulose, and/or lignin.

In one embodiment, the cellulosic-containing material is subjected to abiological pretreatment. The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from thecellulosic-containing material. Biological pretreatment techniques caninvolve applying lignin-solubilizing microorganisms and/or enzymes (see,for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl.Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification and Fermentation of Cellulosic-Containing Material

Saccharification (i.e., hydrolysis) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF).

SHF uses separate process steps to first enzymatically hydrolyze thecellulosic-containing material to fermentable sugars, e.g., glucose,cellobiose, and pentose monomers, and then ferment the fermentablesugars to ethanol. In SSF, the enzymatic hydrolysis of thecellulosic-containing material and the fermentation of sugars to ethanolare combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Biotechnol. Prog. 15: 817-827). HHF involves aseparate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation organism can tolerate. It is understood herein that anymethod known in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the processes described herein.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

In the saccharification step (i.e., hydrolysis step), the cellulosicand/or starch-containing material, e.g., pretreated, is hydrolyzed tobreak down cellulose, hemicellulose, and/or starch to fermentablesugars, such as glucose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. The hydrolysis isperformed enzymatically e.g., by a cellulolytic enzyme composition. Theenzymes of the compositions can be added simultaneously or sequentially.

Enzymatic hydrolysis may be carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic and/or starch-containingmaterial is fed gradually to, for example, an enzyme containinghydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 4.5 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt. %, e.g., about 10 toabout 40 wt. % or about 20 to about 30 wt. %.

Saccharification in may be carried out using a cellulolytic enzymecomposition. Such enzyme compositions are described below in the“Cellulolytic Enzyme Composition’-section below. The cellulolytic enzymecompositions can comprise any protein useful in degrading thecellulosic-containing material. In one aspect, the cellulolytic enzymecomposition comprises or further comprises one or more (e.g., several)proteins selected from the group consisting of a cellulase, an AA9(GH61) polypeptide, a hemicellulase, an esterase, an expansin, aligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and aswollenin.

In another embodiment, the cellulase is preferably one or more (e.g.,several) enzymes selected from the group consisting of an endoglucanase,a cellobiohydrolase, and a beta-glucosidase.

In another embodiment, the hemicellulase is preferably one or more(e.g., several) enzymes selected from the group consisting of anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. In another embodiment, theoxidoreductase is one or more (e.g., several) enzymes selected from thegroup consisting of a catalase, a laccase, and a peroxidase. The enzymesor enzyme compositions used in a processes of the present invention maybe in any form suitable for use, such as, for example, a fermentationbroth formulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

In one embodiment, an effective amount of cellulolytic orhemicellulolytic enzyme composition to the cellulosic-containingmaterial is about 0.5 to about 50 mg, e.g., about 0.5 to about 40 mg,about 0.5 to about 25 mg, about 0.75 to about 20 mg, about 0.75 to about15 mg, about 0.5 to about 10 mg, or about 2.5 to about 10 mg per g ofthe cellulosic-containing material.

In one embodiment, such a compound is added at a molar ratio of thecompound to glucosyl units of cellulose of about 10⁻⁶ to about 10, e.g.,about 10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5,about 10⁻⁶ to about 1, about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹,about 10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹, or about 10⁻³ toabout 10⁻². In another aspect, an effective amount of such a compound isabout 0.1 μM to about 1 M, e.g., about 0.5 μM to about 0.75 M, about0.75 μM to about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about0.1 M, about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50μM to about 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM,or about 0.1 mM to about 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described in WO2012/021401, and the soluble contents thereof. A liquor for cellulolyticenhancement of an AA9 polypeptide (GH61 polypeptide) can be produced bytreating a lignocellulose or hemicellulose material (or feedstock) byapplying heat and/or pressure, optionally in the presence of a catalyst,e.g., acid, optionally in the presence of an organic solvent, andoptionally in combination with physical disruption of the material, andthen separating the solution from the residual solids. Such conditionsdetermine the degree of cellulolytic enhancement obtainable through thecombination of liquor and an AA9 polypeptide during hydrolysis of acellulosic substrate by a cellulolytic enzyme preparation. The liquorcan be separated from the treated material using a method standard inthe art, such as filtration, sedimentation, or centrifugation.

In one embodiment, an effective amount of the liquor to cellulose isabout 10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about7.5 g, about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ toabout 1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about10⁻⁴ to about 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about10⁻² g per g of cellulose.

In the fermentation step, sugars, released from thecellulosic-containing material, e.g., as a result of the pretreatmentand enzymatic hydrolysis steps, are fermented to ethanol, by afermenting organism, such as yeast described herein. Hydrolysis(saccharification) and fermentation can be separate or simultaneous.

Any suitable hydrolyzed cellulosic-containing material can be used inthe fermentation step in practicing the processes described herein. Suchfeedstocks include, but are not limited to carbohydrates (e.g.,lignocellulose, xylans, cellulose, starch, etc.). The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

Production of ethanol by a fermenting organism usingcellulosic-containing material results from the metabolism of sugars(monosaccharides). The sugar composition of the hydrolyzedcellulosic-containing material and the ability of the fermentingorganism to utilize the different sugars has a direct impact in processyields. Prior to Applicant's disclosure herein, strains known in the artutilize glucose efficiently but do not (or very limitedly) metabolizepentoses like xylose, a monosaccharide commonly found in hydrolyzedmaterial.

Compositions of the fermentation media and fermentation conditionsdepend on the fermenting organism and can easily be determined by oneskilled in the art. Typically, the fermentation takes place underconditions known to be suitable for generating the fermentation product.In some embodiments, the fermentation process is carried out underaerobic or microaerophilic (i.e., where the concentration of oxygen isless than that in air), or anaerobic conditions. In some embodiments,fermentation is conducted under anaerobic conditions (i.e., nodetectable oxygen), or less than about 5, about 2.5, or about 1 mmol/L/hoxygen. In the absence of oxygen, the NADH produced in glycolysis cannotbe oxidized by oxidative phosphorylation. Under anaerobic conditions,pyruvate or a derivative thereof may be utilized by the host cell as anelectron and hydrogen acceptor in order to generate NAD+.

The fermentation process is typically run at a temperature that isoptimal for the recombinant fungal cell. For example, in someembodiments, the fermentation process is performed at a temperature inthe range of from about 25° C. to about 42° C. Typically the process iscarried out a temperature that is less than about 38° C., less thanabout 35° C., less than about 33° C., or less than about 38° C., but atleast about 20° C., 22° C., or 25° C.

A fermentation stimulator can be used in a process described herein tofurther improve the fermentation, and in particular, the performance ofthe fermenting organism, such as, rate enhancement and product yield(e.g., ethanol yield). A “fermentation stimulator” refers to stimulatorsfor growth of the fermenting organisms, in particular, yeast. Preferredfermentation stimulators for growth include vitamins and minerals.Examples of vitamins include multivitamins, biotin, pantothenate,nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoicacid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. See, forexample, Alfenore et al., Improving ethanol production and viability ofSaccharomyces cerevisiae by a vitamin feeding strategy during fed-batchprocess, Springer-Verlag (2002), which is hereby incorporated byreference. Examples of minerals include minerals and mineral salts thatcan supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Cellulolytic Enzymes and Compositions

A cellulolytic enzyme or cellulolytic enzyme composition may be presentand/or added during saccharification. A cellulolytic enzyme compositionis an enzyme preparation containing one or more (e.g., several) enzymesthat hydrolyze cellulosic-containing material. Such enzymes includeendoglucanase, cellobiohydrolase, beta-glucosidase, and/or combinationsthereof.

In some embodiments, the fermenting organism comprises one or more(e.g., several) heterologous polynucleotides encoding enzymes thathydrolyze cellulosic-containing material (e.g., an endoglucanase,cellobiohydrolase, beta-glucosidase or combinations thereof). Any enzymedescribed or referenced herein that hydrolyzes cellulosic-containingmaterial is contemplated for expression in the fermenting organism.

The cellulolytic enzyme may be any cellulolytic enzyme that is suitablefor the host cells and/or the methods described herein (e.g., anendoglucanase, cellobiohydrolase, beta-glucosidase), such as a naturallyoccurring cellulolytic enzyme or a variant thereof that retainscellulolytic enzyme activity.

In some embodiments, the fermenting organism comprising a heterologouspolynucleotide encoding a cellulolytic enzyme has an increased level ofcellulolytic enzyme activity (e.g., increased endoglucanase,cellobiohydrolase, and/or beta-glucosidase) compared to the host cellswithout the heterologous polynucleotide encoding the cellulolyticenzyme, when cultivated under the same conditions. In some embodiments,the fermenting organism has an increased level of cellulolytic enzymeactivity of at least 5%, e.g., at least 10%, at least 15%, at least 20%,at least 25%, at least 50%, at least 100%, at least 150%, at least 200%,at least 300%, or at 500% compared to the fermenting organism withoutthe heterologous polynucleotide encoding the cellulolytic enzyme, whencultivated under the same conditions.

Exemplary cellulolytic enzymes that can be used with the host cellsand/or the methods described herein include bacterial, yeast, orfilamentous fungal cellulolytic enzymes, e.g., obtained from any of themicroorganisms described or referenced herein, as described supra underthe sections related to proteases.

The cellulolytic enzyme may be of any origin. In an embodiment thecellulolytic enzyme is derived from a strain of Trichoderma, such as astrain of Trichoderma reesei; a strain of Humicola, such as a strain ofHumicola insolens, and/or a strain of Chrysosporium, such as a strain ofChrysosporium lucknowense. In a preferred embodiment the cellulolyticenzyme is derived from a strain of Trichoderma reesei.

The cellulolytic enzyme composition may further comprise one or more ofthe following polypeptides, such as enzymes: AA9 polypeptide (GH61polypeptide) having cellulolytic enhancing activity, beta-glucosidase,xylanase, beta-xylosidase, CBH I, CBH II, or a mixture of two, three,four, five or six thereof.

The further polypeptide(s) (e.g., AA9 polypeptide) and/or enzyme(s)(e.g., beta-glucosidase, xylanase, beta-xylosidase, CBH I and/or CBH IImay be foreign to the cellulolytic enzyme composition producing organism(e.g., Trichoderma reesei).

In an embodiment the cellulolytic enzyme composition comprises an AA9polypeptide having cellulolytic enhancing activity and abeta-glucosidase.

In another embodiment the cellulolytic enzyme composition comprises anAA9 polypeptide having cellulolytic enhancing activity, abeta-glucosidase, and a CBH I.

In another embodiment the cellulolytic enzyme composition comprises anAA9 polypeptide having cellulolytic enhancing activity, abeta-glucosidase, a CBH I and a CBH II. Other enzymes, such asendoglucanases, may also be comprised in the cellulolytic enzymecomposition.

As mentioned above the cellulolytic enzyme composition may comprise anumber of difference polypeptides, including enzymes.

In one embodiment, the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition, further comprising Thermoascusaurantiacus AA9 (GH61A) polypeptide having cellulolytic enhancingactivity (e.g., WO 2005/074656), and Aspergillus oryzae beta-glucosidasefusion protein (e.g., one disclosed in WO 2008/057637, in particularshown as SEQ ID NOs: 59 and 60).

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition, further comprisingThermoascus aurantiacus AA9 (GH61A) polypeptide having cellulolyticenhancing activity (e.g., SEQ ID NO: 2 in WO 2005/074656), andAspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO2005/047499).

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition, further comprisingPenicillium emersonii AA9 (GH61A) polypeptide having cellulolyticenhancing activity, in particular the one disclosed in WO 2011/041397,and Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO2005/047499).

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition, further comprisingPenicillium emersonii AA9 (GH61A) polypeptide having cellulolyticenhancing activity, in particular the one disclosed in WO 2011/041397,and Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO2005/047499) or a variant disclosed in WO 2012/044915 (herebyincorporated by reference), in particular one comprising one or moresuch as all of the following substitutions: F100D, S283G, N456E, F512Y.

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic composition, further comprising an AA9 (GH61A)polypeptide having cellulolytic enhancing activity, in particular theone derived from a strain of Penicillium emersonii (e.g., SEQ ID NO: 2in WO 2011/041397), Aspergillus fumigatus beta-glucosidase (e.g., SEQ IDNO: 2 in WO 2005/047499) variant with one or more, in particular all ofthe following substitutions: F100D, S283G, N456E, F512Y and disclosed inWO 2012/044915; Aspergillus fumigatus Cel7A CBH1, e.g., the onedisclosed as SEQ ID NO: 6 in WO2011/057140 and Aspergillus fumigatus CBHII, e.g., the one disclosed as SEQ ID NO: 18 in WO 2011/057140.

In a preferred embodiment the cellulolytic enzyme composition is aTrichoderma reesei, cellulolytic enzyme composition, further comprisinga hemicellulase or hemicellulolytic enzyme composition, such as anAspergillus fumigatus xylanase and Aspergillus fumigatusbeta-xylosidase.

In an embodiment the cellulolytic enzyme composition also comprises axylanase (e.g., derived from a strain of the genus Aspergillus, inparticular Aspergillus aculeatus or Aspergillus fumigatus; or a strainof the genus Talaromyces, in particular Talaromyces leycettanus) and/ora beta-xylosidase (e.g., derived from Aspergillus, in particularAspergillus fumigatus, or a strain of Talaromyces, in particularTalaromyces emersonii).

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition, further comprising Thermoascusaurantiacus AA9 (GH61A) polypeptide having cellulolytic enhancingactivity (e.g., WO 2005/074656), Aspergillus oryzae beta-glucosidasefusion protein (e.g., one disclosed in WO 2008/057637, in particular asSEQ ID NOs: 59 and 60), and Aspergillus aculeatus xylanase (e.g., Xyl IIin WO 94/21785).

In another embodiment the cellulolytic enzyme composition comprises aTrichoderma reesei cellulolytic preparation, further comprisingThermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity (e.g., SEQ ID NO: 2 in WO 2005/074656), Aspergillus fumigatusbeta-glucosidase (e.g., SEQ ID NO: 2 of WO 2005/047499) and Aspergillusaculeatus xylanase (Xyl II disclosed in WO 94/21785).

In another embodiment the cellulolytic enzyme composition comprises aTrichoderma reesei cellulolytic enzyme composition, further comprisingThermoascus aurantiacus AA9 (GH61A) polypeptide having cellulolyticenhancing activity (e.g., SEQ ID NO: 2 in WO 2005/074656), Aspergillusfumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO 2005/047499) andAspergillus aculeatus xylanase (e.g., Xyl II disclosed in WO 94/21785).

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition, further comprisingPenicillium emersonii AA9 (GH61A) polypeptide having cellulolyticenhancing activity, in particular the one disclosed in WO 2011/041397,Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO2005/047499) and Aspergillus fumigatus xylanase (e.g., Xyl III in WO2006/078256).

In another embodiment the cellulolytic enzyme composition comprises aTrichoderma reesei cellulolytic enzyme composition, further comprisingPenicillium emersonii AA9 (GH61A) polypeptide having cellulolyticenhancing activity, in particular the one disclosed in WO 2011/041397,Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO2005/047499), Aspergillus fumigatus xylanase (e.g., Xyl III in WO2006/078256), and CBH I from Aspergillus fumigatus, in particular Cel7ACBH1 disclosed as SEQ ID NO: 2 in WO2011/057140.

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition, further comprisingPenicillium emersonii AA9 (GH61A) polypeptide having cellulolyticenhancing activity, in particular the one disclosed in WO 2011/041397,Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO2005/047499), Aspergillus fumigatus xylanase (e.g., Xyl III in WO2006/078256), CBH I from Aspergillus fumigatus, in particular Cel7A CBH1disclosed as SEQ ID NO: 2 in WO 2011/057140, and CBH II derived fromAspergillus fumigatus in particular the one disclosed as SEQ ID NO: 4 inWO 2013/028928.

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition, further comprisingPenicillium emersonii AA9 (GH61A) polypeptide having cellulolyticenhancing activity, in particular the one disclosed in WO 2011/041397,Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO2005/047499) or variant thereof with one or more, in particular all, ofthe following substitutions: F100D, S283G, N456E, F512Y; Aspergillusfumigatus xylanase (e.g., Xyl III in WO 2006/078256), CBH I fromAspergillus fumigatus, in particular Cel7A CBH I disclosed as SEQ ID NO:2 in WO 2011/057140, and CBH II derived from Aspergillus fumigatus, inparticular the one disclosed in WO 2013/028928.

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition comprising the CBH I(GENSEQP Accession No. AZY49536 (WO2012/103293); a CBH II (GENSEQPAccession No. AZY49446 (WO2012/103288); a beta-glucosidase variant(GENSEQP Accession No. AZU67153 (WO 2012/44915)), in particular with oneor more, in particular all, of the following substitutions: F100D,S283G, N456E, F512Y; and AA9 (GH61 polypeptide) (GENSEQP Accession No.BAL61510 (WO 2013/028912)).

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition comprising a CBH I(GENSEQP Accession No. AZY49536 (WO2012/103293)); a CBH II (GENSEQPAccession No. AZY49446 (WO2012/103288); a GH10 xylanase (GENSEQPAccession No. BAK46118 (WO 2013/019827)); and a beta-xylosidase (GENSEQPAccession No. AZI04896 (WO 2011/057140)).

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition comprising a CBH I(GENSEQP Accession No. AZY49536 (WO2012/103293)); a CBH II (GENSEQPAccession No. AZY49446 (WO2012/103288)); and an AA9 (GH61 polypeptide;GENSEQP Accession No. BAL61510 (WO 2013/028912)).

In another embodiment the cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition comprising a CBH I(GENSEQP Accession No. AZY49536 (WO2012/103293)); a CBH II (GENSEQPAccession No. AZY49446 (WO2012/103288)), an AA9 (GH61 polypeptide;GENSEQP Accession No. BAL61510 (WO 2013/028912)), and a catalase(GENSEQP Accession No. BAC11005 (WO 2012/130120)).

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition comprising a CBH I (GENSEQPAccession No. AZY49446 (WO2012/103288); a CBH II (GENSEQP Accession No.AZY49446 (WO2012/103288)), a beta-glucosidase variant (GENSEQP AccessionNo. AZU67153 (WO 2012/44915)), with one or more, in particular all, ofthe following substitutions: F100D, S283G, N456E, F512Y; an AA9 (GH61polypeptide; GENSEQP Accession No. BAL61510 (WO 2013/028912)), a GH10xylanase (GENSEQP Accession No. BAK46118 (WO 2013/019827)), and abeta-xylosidase (GENSEQP Accession No. AZI04896 (WO 2011/057140)).

In an embodiment the cellulolytic composition is a Trichoderma reeseicellulolytic enzyme preparation comprising an EG I (Swissprot AccessionNo. P07981), EG 11 (EMBL Accession No. M19373), CBH I (supra); CBH II(supra); beta-glucosidase variant (supra) with the followingsubstitutions: F100D, S283G, N456E, F512Y; an AA9 (GH61 polypeptide;supra), GH10 xylanase (supra); and beta-xylosidase (supra).

All cellulolytic enzyme compositions disclosed in WO 2013/028928 arealso contemplated and hereby incorporated by reference.

The cellulolytic enzyme composition comprises or may further compriseone or more (several) proteins selected from the group consisting of acellulase, a AA9 (i.e., GH61) polypeptide having cellulolytic enhancingactivity, a hemicellulase, an expansin, an esterase, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

In one embodiment the cellulolytic enzyme composition is a commercialcellulolytic enzyme composition. Examples of commercial cellulolyticenzyme compositions suitable for use in a process of the inventioninclude: CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S),CELLIC® CTec3 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), SPEZYME™ CP(Genencor Int.), ACCELLERASE™ 1000, ACCELLERASE 1500, ACCELLERASE™ TRIO(DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W(Röhm GmbH), or ALTERNAFUEL® CMAX3™ (Dyadic International, Inc.). Thecellulolytic enzyme composition may be added in an amount effective fromabout 0.001 to about 5.0 wt. % of solids, e.g., about 0.025 to about 4.0wt. % of solids or about 0.005 to about 2.0 wt. % of solids.

Additional enzymes, and compositions thereof can be found inWO2011/153516 and WO2016/045569 (the contents of which are incorporatedherein).

Additional polynucleotides encoding suitable cellulolytic enzymes may beobtained from microorganisms of any genus, including those readilyavailable within the UniProtKB database (www.uniprot.org).

The cellulolytic enzyme coding sequences can also be used to designnucleic acid probes to identify and clone DNA encoding cellulolyticenzymes from strains of different genera or species, as described supra.

The polynucleotides encoding cellulolytic enzymes may also be identifiedand obtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encodingcellulolytic enzymes are described supra.

In one embodiment, the cellulolytic enzyme has a mature polypeptidesequence of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to anycellulolytic enzyme described or referenced herein (e.g., anyendoglucanase, cellobiohydrolase, or beta-glucosidase). In one aspect,the cellulolytic enzyme ha a mature polypeptide sequence that differs byno more than ten amino acids, e.g., by no more than five amino acids, byno more than four amino acids, by no more than three amino acids, by nomore than two amino acids, or by one amino acid from any cellulolyticenzyme described or referenced herein. In one embodiment, thecellulolytic enzyme has a mature polypeptide sequence that comprises orconsists of the amino acid sequence of any cellulolytic enzyme describedor referenced herein, allelic variant, or a fragment thereof havingcellulolytic enzyme activity. In one embodiment, the cellulolytic enzymehas an amino acid substitution, deletion, and/or insertion of one ormore (e.g., two, several) amino acids. In some embodiments, the totalnumber of amino acid substitutions, deletions and/or insertions is notmore than 10, e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1.

In some embodiments, the cellulolytic enzyme has at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% of the cellulolytic enzyme activity of anycellulolytic enzyme described or referenced herein (e.g., anyendoglucanase, cellobiohydrolase, or beta-glucosidase) under the sameconditions.

In one embodiment, the cellulolytic enzyme coding sequence hybridizesunder at least low stringency conditions, e.g., medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the full-lengthcomplementary strand of the coding sequence from any cellulolytic enzymedescribed or referenced herein (e.g., any endoglucanase,cellobiohydrolase, or beta-glucosidase). In one embodiment, thecellulolytic enzyme coding sequence has at least 65%, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the coding sequence from any cellulolytic enzymedescribed or referenced herein.

In one embodiment, the polynucleotide encoding the cellulolytic enzymecomprises the coding sequence of any cellulolytic enzyme described orreferenced herein (e.g., any endoglucanase, cellobiohydrolase, orbeta-glucosidase). In one embodiment, the polynucleotide encoding thecellulolytic enzyme comprises a subsequence of the coding sequence fromany cellulolytic enzyme described or referenced herein, wherein thesubsequence encodes a polypeptide having cellulolytic enzyme activity.In one embodiment, the number of nucleotides residues in the subsequenceis at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number ofthe referenced coding sequence.

The cellulolytic enzyme can also include fused polypeptides or cleavablefusion polypeptides, as described supra.

Xylose Metabolism

In one aspect, the fermenting organism (e.g., yeast cell) furthercomprises a heterologous polynucleotide encoding a xylose isomerase(XI). The xylose isomerase may be any xylose isomerase that is suitablefor the host cells and the methods described herein, such as a naturallyoccurring xylose isomerase or a variant thereof that retains xyloseisomerase activity. In one embodiment, the xylose isomerase is presentin the cytosol of the host cells.

In some embodiments, the fermenting organism comprising a heterologouspolynucleotide encoding a xylose isomerase has an increased level ofxylose isomerase activity compared to the host cells without theheterologous polynucleotide encoding the xylose isomerase, whencultivated under the same conditions. In some embodiments, thefermenting organisms have an increased level of xylose isomeraseactivity of at least 5%, e.g., at least 10%, at least 15%, at least 20%,at least 25%, at least 50%, at least 100%, at least 150%, at least 200%,at least 300%, or at 500% compared to the host cells without theheterologous polynucleotide encoding the xylose isomerase, whencultivated under the same conditions.

Exemplary xylose isomerases that can be used with the recombinant hostcells and methods of use described herein include, but are not limitedto, Xls from the fungus Piromyces sp. (WO2003/062430) or other sources(Madhavan et al., 2009, Appl Microbiol Biotechnol. 82(6), 1067-1078)have been expressed in S. cerevisiae host cells. Still other Xlssuitable for expression in yeast have been described in US 2012/0184020(an XI from Ruminococcus flavefaciens), WO2011/078262 (several Xls fromReticulitermes speratus and Mastotermes darwiniensis) and WO2012/009272(constructs and fungal cells containing an XI from Abiotrophiadefectiva). U.S. Pat. No. 8,586,336 describes a S. cerevisiae host cellexpressing an XI obtained by bovine rumen fluid (shown herein as SEQ IDNO: 74).

Additional polynucleotides encoding suitable xylose isomerases may beobtained from microorganisms of any genus, including those readilyavailable within the UniProtKB database (www.uniprot.org). In oneembodiment, the xylose isomerases is a bacterial, a yeast, or afilamentous fungal xylose isomerase, e.g., obtained from any of themicroorganisms described or referenced herein, as described supra.

The xylose isomerase coding sequences can also be used to design nucleicacid probes to identify and clone DNA encoding xylose isomerases fromstrains of different genera or species, as described supra.

The polynucleotides encoding xylose isomerases may also be identifiedand obtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encoding xyloseisomerases are described supra.

In one embodiment, the xylose isomerase has a mature polypeptidesequence of having at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to anyxylose isomerase described or referenced herein (e.g., the xyloseisomerase of SEQ ID NO: 74). In one aspect, the xylose isomerase has amature polypeptide sequence that differs by no more than ten aminoacids, e.g., by no more than five amino acids, by no more than fouramino acids, by no more than three amino acids, by no more than twoamino acids, or by one amino acid from any xylose isomerase described orreferenced herein (e.g., the xylose isomerase of SEQ ID NO: 74). In oneembodiment, the xylose isomerase has a mature polypeptide sequence thatcomprises or consists of the amino acid sequence of any xylose isomerasedescribed or referenced herein (e.g., the xylose isomerase of SEQ ID NO:74), allelic variant, or a fragment thereof having xylose isomeraseactivity. In one embodiment, the xylose isomerase has an amino acidsubstitution, deletion, and/or insertion of one or more (e.g., two,several) amino acids. In some embodiments, the total number of aminoacid substitutions, deletions and/or insertions is not more than 10,e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1.

In some embodiments, the xylose isomerase has at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% of the xylose isomerase activity of any xyloseisomerase described or referenced herein (e.g., the xylose isomerase ofSEQ ID NO: 74) under the same conditions.

In one embodiment, the xylose isomerase coding sequence hybridizes underat least low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any xylose isomerase described or referencedherein (e.g., the xylose isomerase of SEQ ID NO: 74). In one embodiment,the xylose isomerase coding sequence has at least 65%, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the coding sequence from any xylose isomerasedescribed or referenced herein (e.g., the xylose isomerase of SEQ ID NO:74).

In one embodiment, the heterologous polynucleotide encoding the xyloseisomerase comprises the coding sequence of any xylose isomerasedescribed or referenced herein (e.g., the xylose isomerase of SEQ ID NO:74). In one embodiment, the heterologous polynucleotide encoding thexylose isomerase comprises a subsequence of the coding sequence from anyxylose isomerase described or referenced herein, wherein the subsequenceencodes a polypeptide having xylose isomerase activity. In oneembodiment, the number of nucleotides residues in the subsequence is atleast 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of thereferenced coding sequence.

The xylose isomerases can also include fused polypeptides or cleavablefusion polypeptides, as described supra.

In one aspect, the fermenting organism (e.g., yeast cell) furthercomprises a heterologous polynucleotide encoding a xylulokinase (XK). Axylulokinase, as used herein, provides enzymatic activity for convertingD-xylulose to xylulose 5-phosphate. The xylulokinase may be anyxylulokinase that is suitable for the host cells and the methodsdescribed herein, such as a naturally occurring xylulokinase or avariant thereof that retains xylulokinase activity. In one embodiment,the xylulokinase is present in the cytosol of the host cells.

In some embodiments, the fermenting organisms comprising a heterologouspolynucleotide encoding a xylulokinase have an increased level ofxylulokinase activity compared to the host cells without theheterologous polynucleotide encoding the xylulokinase, when cultivatedunder the same conditions. In some embodiments, the host cells have anincreased level of xylose isomerase activity of at least 5%, e.g., atleast 10%, at least 15%, at least 20%, at least 25%, at least 50%, atleast 100%, at least 150%, at least 200%, at least 300%, or at 500%compared to the host cells without the heterologous polynucleotideencoding the xylulokinase, when cultivated under the same conditions.

Exemplary xylulokinases that can be used with the fermenting organismsand methods of use described herein include, but are not limited to, theSaccharomyces cerevisiae xylulokinase of SEQ ID NO: 75. Additionalpolynucleotides encoding suitable xylulokinases may be obtained frommicroorganisms of any genus, including those readily available withinthe UniProtKB database (www.uniprot.org). In one embodiment, thexylulokinases is a bacterial, a yeast, or a filamentous fungalxylulokinase, e.g., obtained from any of the microorganisms described orreferenced herein, as described supra.

The xylulokinase coding sequences can also be used to design nucleicacid probes to identify and clone DNA encoding xylulokinases fromstrains of different genera or species, as described supra.

The polynucleotides encoding xylulokinases may also be identified andobtained from other sources including microorganisms isolated fromnature (e.g., soil, composts, water, etc.) or DNA samples obtaineddirectly from natural materials (e.g., soil, composts, water, etc) asdescribed supra.

Techniques used to isolate or clone polynucleotides encodingxylulokinases are described supra.

In one embodiment, the xylulokinase has a mature polypeptide sequence ofat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to any xylulokinasedescribed or referenced herein (e.g., the Saccharomyces cerevisiaexylulokinase of SEQ ID NO: 75). In one embodiment, the xylulokinase hasa mature polypeptide sequence that differs by no more than ten aminoacids, e.g., by no more than five amino acids, by no more than fouramino acids, by no more than three amino acids, by no more than twoamino acids, or by one amino acid from any xylulokinase described orreferenced herein (e.g., the Saccharomyces cerevisiae xylulokinase ofSEQ ID NO: 75). In one embodiment, the xylulokinase has a maturepolypeptide sequence that comprises or consists of the amino acidsequence of any xylulokinase described or referenced herein (e.g., theSaccharomyces cerevisiae xylulokinase of SEQ ID NO: 75), allelicvariant, or a fragment thereof having xylulokinase activity. In oneembodiment, the xylulokinase has an amino acid substitution, deletion,and/or insertion of one or more (e.g., two, several) amino acids. Insome embodiments, the total number of amino acid substitutions,deletions and/or insertions is not more than 10, e.g., not more than 9,8, 7, 6, 5, 4, 3, 2, or 1.

In some embodiments, the xylulokinase has at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the xylulokinase activity of any xylulokinase describedor referenced herein (e.g., the Saccharomyces cerevisiae xylulokinase ofSEQ ID NO: 75) under the same conditions.

In one embodiment, the xylulokinase coding sequence hybridizes under atleast low stringency conditions, e.g., medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with the full-length complementary strand ofthe coding sequence from any xylulokinase described or referenced herein(e.g., the Saccharomyces cerevisiae xylulokinase of SEQ ID NO: 75). Inone embodiment, the xylulokinase coding sequence has at least 65%, e.g.,at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the coding sequence from any xylulokinasedescribed or referenced herein (e.g., the Saccharomyces cerevisiaexylulokinase of SEQ ID NO: 75).

In one embodiment, the heterologous polynucleotide encoding thexylulokinase comprises the coding sequence of any xylulokinase describedor referenced herein (e.g., the Saccharomyces cerevisiae xylulokinase ofSEQ ID NO: 75). In one embodiment, the heterologous polynucleotideencoding the xylulokinase comprises a subsequence of the coding sequencefrom any xylulokinase described or referenced herein, wherein thesubsequence encodes a polypeptide having xylulokinase activity. In oneembodiment, the number of nucleotides residues in the subsequence is atleast 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of thereferenced coding sequence.

The xylulokinases can also include fused polypeptides or cleavablefusion polypeptides, as described supra.

In one aspect, the fermenting organism (e.g., yeast cell) furthercomprises a heterologous polynucleotide encoding a ribulose 5 phosphate3-epimerase (RPE1). A ribulose 5 phosphate 3-epimerase, as used herein,provides enzymatic activity for converting L-ribulose 5-phosphate toL-xylulose 5-phosphate (EC 5.1.3.22). The RPE1 may be any RPE1 that issuitable for the host cells and the methods described herein, such as anaturally occurring RPE1 or a variant thereof that retains RPE1activity. In one embodiment, the RPE1 is present in the cytosol of thehost cells. In one embodiment, the recombinant cell comprises aheterologous polynucleotide encoding a ribulose 5 phosphate 3-epimerase(RPE1), wherein the RPE1 is Saccharomyces cerevisiae RPE1, or an RPE1having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% sequence identity to a Saccharomyces cerevisiaeRPE1.

In one aspect, the fermenting organism (e.g., yeast cell) furthercomprises a heterologous polynucleotide encoding a ribulose 5 phosphateisomerase (RKI1). A ribulose 5 phosphate isomerase, as used herein,provides enzymatic activity for converting ribose-5-phosphate toribulose 5-phosphate. The RKI1 may be any RKI1 that is suitable for thehost cells and the methods described herein, such as a naturallyoccurring RKI1 or a variant thereof that retains RKI1 activity. In oneembodiment, the RKI1 is present in the cytosol of the host cells.

In one embodiment, the fermenting organism comprises a heterologouspolynucleotide encoding a ribulose 5 phosphate isomerase (RKI1), whereinthe RKI1 is a Saccharomyces cerevisiae RKI1, or an RKI1 having a maturepolypeptide sequence of at least 60%, e.g., at least 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to aSaccharomyces cerevisiae RKI1.

In one aspect, the fermenting organism (e.g., yeast cell) furthercomprises a heterologous polynucleotide encoding a transketolase (TKL1).The TKL1 may be any TKL1 that is suitable for the host cells and themethods described herein, such as a naturally occurring TKL1 or avariant thereof that retains TKL1 activity. In one embodiment, the TKL1is present in the cytosol of the host cells.

In one embodiment, the fermenting organism comprises a heterologouspolynucleotide encoding a transketolase (TKL1), wherein the TKL1 is aSaccharomyces cerevisiae TKL1, or a TKL1 having a mature polypeptidesequence of at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100% sequence identity to a Saccharomycescerevisiae TKL1.

In one aspect, the fermenting organism (e.g., yeast cell) furthercomprises a heterologous polynucleotide encoding a transaldolase (TAL1).The TAL1 may be any TAL1 that is suitable for the host cells and themethods described herein, such as a naturally occurring TAL1 or avariant thereof that retains TAL1 activity. In one embodiment, the TAL1is present in the cytosol of the host cells.

In one embodiment, the fermenting organism comprises a heterologouspolynucleotide encoding a transketolase (TAL1), wherein the TAL1 is aSaccharomyces cerevisiae TAL1, or a TAL1 having a mature polypeptidesequence of at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100% sequence identity to a Saccharomycescerevisiae TAL1.

Fermentation Products

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g., pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide.

In one aspect, the fermentation product is an alcohol. The term“alcohol” encompasses a substance that contains one or more hydroxylmoieties. The alcohol can be, but is not limited to, n-butanol,isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol,glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol. See, forexample, Gong et al., 1999, Ethanol production from renewable resources,in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira andJonas, 2002, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh,1995, Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, WorldJournal of Microbiology and Biotechnology 19(6): 595-603. In oneembodiment, the fermentation product is ethanol.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene. In another aspect, thefermentation product is an amino acid. The organic acid can be, but isnot limited to, aspartic acid, glutamic acid, glycine, lysine, serine,or threonine. See, for example, Richard and Margaritis, 2004,Biotechnology and Bioengineering 87(4): 501-515.

In another aspect, the fermentation product is a gas. The gas can be,but is not limited to, methane, H₂, CO₂, or CO. See, for example,Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; andGunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. The term“ketone” encompasses a substance that contains one or more ketonemoieties. The ketone can be, but is not limited to, acetone.

In another aspect, the fermentation product is an organic acid. Theorganic acid can be, but is not limited to, acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, propionic acid, succinicacid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

In some aspects, the fermenting organism (or processes thereof), providehigher yield of fermentation product (e.g., ethanol) when compared tothe same process using an identical cell without the heterologouspolynucleotide encoding the phospholipase under the same conditions(e.g., at about or after 54 hours fermentation, such as the conditionsdescribed in Example 3 or 4). In some embodiments, the process resultsin at least 0.25%, such as 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2%, 3%or 5% higher yield of the fermentation product (e.g., ethanol).

Recovery

The fermentation product, e.g., ethanol, can optionally be recoveredfrom the fermentation medium using any method known in the artincluding, but not limited to, chromatography, electrophoreticprocedures, differential solubility, distillation, or extraction. Forexample, alcohol is separated from the fermented cellulosic material andpurified by conventional methods of distillation. Ethanol with a purityof up to about 96 vol. % can be obtained, which can be used as, forexample, fuel ethanol, drinking ethanol, i.e., potable neutral spirits,or industrial ethanol.

In some aspects of the methods, the fermentation product after beingrecovered is substantially pure. With respect to the methods herein,“substantially pure” intends a recovered preparation that contains nomore than 15% impurity, wherein impurity intends compounds other thanthe fermentation product (e.g., ethanol). In one variation, asubstantially pure preparation is provided wherein the preparationcontains no more than 25% impurity, or no more than 20% impurity, or nomore than 10% impurity, or no more than 5% impurity, or no more than 3%impurity, or no more than 1% impurity, or no more than 0.5% impurity.

Suitable assays to test for the production of ethanol and contaminants,and sugar consumption can be performed using methods known in the art.For example, ethanol product, as well as other organic compounds, can beanalyzed by methods such as HPLC (High Performance LiquidChromatography), GC-MS (Gas Chromatography Mass Spectroscopy) and LC-MS(Liquid Chromatography-Mass Spectroscopy) or other suitable analyticalmethods using routine procedures well known in the art. The release ofethanol in the fermentation broth can also be tested with the culturesupernatant. Byproducts and residual sugar in the fermentation medium(e.g., glucose or xylose) can be quantified by HPLC using, for example,a refractive index detector for glucose and alcohols, and a UV detectorfor organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)),or using other suitable assay and detection methods well known in theart.

The invention may further be described in the following numberedparagraphs:

Paragraph [1]. A method of producing a fermentation product from astarch-containing or cellulosic-containing material comprising:(a) saccharifying the starch-containing or cellulosic-containingmaterial; and(b) fermenting the saccharified material of step (a) with a fermentingorganism;

wherein the fermenting organism comprises a heterologous polynucleotideencoding a phospholipase.

Paragraph [2]. The method of paragraph [1], wherein the phospholipase isa Phospholipase A or a Phospholipase C.Paragraph [3]. The method of paragraph [1] or [2], wherein thephospholipase has a mature polypeptide sequence with 60%, e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity, to the amino acid sequence of any one of SEQ ID NOs: 235-242and 252-342 (e.g., any one of SEQ ID NOs: 235, 236, 237, 238, 239, 240,241 and 242).Paragraph [4]. The method of any one of paragraphs [1]-[3], wherein theheterologous polynucleotide encodes a phospholipase having a maturepolypeptide sequence that differs by no more than ten amino acids, e.g.,by no more than five amino acids, by no more than four amino acids, byno more than three amino acids, by no more than two amino acids, or byone amino acid from the amino acid sequence of any one of SEQ ID NOs:235-242 and 252-342 (e.g., any one of SEQ ID NOs: 235, 236, 237, 238,239, 240, 241 and 242).Paragraph [5]. The method of any one of paragraphs [1]-[4], wherein theheterologous polynucleotide encodes a phospholipase having a maturepolypeptide sequence comprising or consisting of the amino acid sequenceof any one of SEQ ID NOs: 235-242 and 252-342 (e.g., any one of SEQ IDNOs: 235, 236, 237, 238, 239, 240, 241 and 242).Paragraph [6]. The method of any one of paragraphs [1]-[5], whereinsaccharification of step (a) occurs on a starch-containing material, andwherein the starch-containing material is either gelatinized orungelatinized starch.Paragraph [7]. The method of paragraph [6], comprising liquefying thestarch-containing material by contacting the material with analpha-amylase prior to saccharification.Paragraph [8]. The method of paragraph [7], wherein liquefying thestarch-containing material and/or saccharifying the starch-containingmaterial is conducted in presence of exogenously added protease.Paragraph [9]. The method of any one of paragraphs [1]-[8], whereinfermentation is performed under reduced nitrogen conditions (e.g., lessthan 1000 ppm urea or ammonium hydroxide, such as less than 750 ppm,less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 250ppm, less than 200 ppm, less than 150 ppm, less than 100 ppm, less than75 ppm, less than 50 ppm, less than 25 ppm, or less than 10 ppm).Paragraph [10]. The method of any one of paragraphs [1]-[9], whereinfermentation and saccharification are performed simultaneously in asimultaneous saccharification and fermentation (SSF).Paragraph [11]. The method of any one of paragraphs [1]-[9], whereinfermentation and saccharification are performed sequentially (SHF).Paragraph [12]. The method of any one of paragraphs paragraph [1]-[11],comprising recovering the fermentation product from the fermentation.Paragraph [13]. The method of paragraph [12], wherein recovering thefermentation product from the from the fermentation comprisesdistillation.Paragraph [14]. The method of any one of paragraphs [1]-[13], whereinthe fermentation product is ethanol.Paragraph [15]. The method of any one of paragraphs [1]-[14], whereinthe fermenting organism comprises a heterologous polynucleotide encodinga glucoamylase.Paragraph [16]. The method of paragraph [15], wherein the glucoamylasehas a mature polypeptide sequence with 60%, e.g., at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity, tothe amino acid sequence of a Pycnoporus glycoamylase (e.g., a Pycnoporussanguineus glucoamylase of SEQ ID NO: 229), a Gloeophyllum glucoamylase(e.g. a Gloeophyllum sepiarium of SEQ ID NO: 8), or a glucoamylase ofany one of SEQ ID NOs: 102-113 (e.g., a Saccharomycopsis fibuligeraglucoamylase of SEQ ID NO: 103 or 104, or a Trichoderma reeseiglucoamylase of SEQ ID NO: 230).Paragraph [17]. The method of any one of paragraphs [1]-[16], whereinthe fermenting organism comprises a heterologous polynucleotide encodingan alpha-amylase.Paragraph [18]. The method of paragraph [17], wherein the alpha-amylasehas a mature polypeptide sequence of at least 60%, e.g., at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identityto the amino acid sequence of any one of SEQ ID NOs: 76-101, 121-174 and231.Paragraph [19]. The method of any one of paragraphs [1]-[18], whereinthe fermenting organism comprises a heterologous polynucleotide encodinga protease.Paragraph [20]. The method of paragraph [19], wherein the protease has amature polypeptide sequence of at least 60%, e.g., at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of any one of SEQ ID NOs: 9-73 (e.g., any one of SEQID NOs: 9, 14, 16, 21, 22, 33, 41, 45, 61, 62, 66, 67, and 69; such asany one of SEQ NOs: 9, 14, 16, and 69).Paragraph [21]. The method of any one of paragraphs [1]-[20], whereinthe fermenting organism comprises a heterologous polynucleotide encodinga trehalase.Paragraph [22]. The method of paragraph [21], wherein the trehalase hasa mature polypeptide sequence of at least 60%, e.g., at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of any one of SEQ ID NOs: 175-226.Paragraph [23]. The method of any one of paragraphs [1]-[22], whereinsaccharification of step occurs on a cellulosic-containing material, andwherein the cellulosic-containing material is pretreated.Paragraph [24]. The method of paragraph [23], wherein the pretreatmentis a dilute acid pretreatment.Paragraph [25]. The method of any one of paragraphs [1]-[24], whereinsaccharification occurs on a cellulosic-containing material, and whereinthe enzyme composition comprises one or more enzymes selected from acellulase, an AA9 polypeptide, a hemicellulase, a CIP, an esterase, anexpansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, aprotease, and a swollenin.Paragraph [26]. The method of paragraph [25], wherein the cellulase isone or more enzymes selected from an endoglucanase, a cellobiohydrolase,and a beta-glucosidase.Paragraph [27]. The method of paragraph [25] or [26], wherein thehemicellulase is one or more enzymes selected a xylanase, an acetylxylanesterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, anda glucuronidase.Paragraph [28]. The method of any one of paragraphs [1]-[27], whereinthe fermenting organism is a Saccharomyces, Rhodotorula,Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Rhodosporidium,Candida, Yarrowia, Lipomyces, Cryptococcus, or Dekkera sp. cell.Paragraph [29]. The method of any one of paragraphs [1]-[28], whereinthe fermenting organism is a Saccharomyces cerevisiae cell.Paragraph [30]. The method of any one of paragraphs [1]-[29], whereinthe method results in higher yield of fermentation product and/orreduced foam accumulation when compared to the same process using anidentical cell without the heterologous polynucleotide encoding thephospholipase under the same conditions (e.g., at about or after 54hours fermentation, such as the conditions described in Examples 3 or4).Paragraph [31]. The method of paragraph [30], wherein the method resultsin at least 0.25% (e.g., 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2%, 3%or 5%) higher yield of fermentation product.Paragraph [32]. A recombinant yeast cell comprising a heterologouspolynucleotide encoding a phospholipase.Paragraph [33]. The recombinant yeast cell of paragraph [32], whereinthe phospholipase is a Phospholipase A or a Phospholipase C.Paragraph [34]. The recombinant yeast cell of paragraph [32] or [33],wherein the phospholipase has a mature polypeptide sequence with 60%,e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%sequence identity, to the amino acid sequence of any one of SEQ ID NOs:235-242 and 252-342 (e.g., any one of SEQ ID NOs: 235, 236, 237, 238,239, 240, 241 and 242).Paragraph [35]. The recombinant yeast cell of any one of paragraphs[32]-[34], wherein the heterologous polynucleotide encodes aphospholipase having a mature polypeptide sequence that differs by nomore than ten amino acids, e.g., by no more than five amino acids, by nomore than four amino acids, by no more than three amino acids, by nomore than two amino acids, or by one amino acid from the amino acidsequence of any one of SEQ ID NOs: 235-242 and 252-342 (e.g., any one ofSEQ ID NOs: 235, 236, 237, 238, 239, 240, 241 and 242).Paragraph [36]. The recombinant yeast cell of any one of paragraphs[32]-[35], wherein the heterologous polynucleotide encodes aphospholipase having a mature polypeptide sequence comprising orconsisting of the amino acid sequence of any one of SEQ ID NOs: 235-242and 252-342 (e.g., any one of SEQ ID NOs: 235, 236, 237, 238, 239, 240,241 and 242.Paragraph [37]. The recombinant yeast cell of any one of paragraphs[32]-[36], wherein the fermenting organism comprises a heterologouspolynucleotide encoding a glucoamylase.Paragraph [38]. The recombinant yeast cell of paragraph [37], whereinthe glucoamylase has a mature polypeptide sequence with 60%, e.g., atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity, to the amino acid sequence of a Pycnoporus glycoamylase (e.g.,a Pycnoporus sanguineus glucoamylase of SEQ ID NO: 229), a Gloeophyllumglucoamylase (e.g. a Gloeophyllum sepiarium of SEQ ID NO: 8), or aglucoamylase of any one of SEQ ID NOs: 102-113 (e.g., a Saccharomycopsisfibuligera glucoamylase of SEQ ID NO: 103 or 104, or a Trichodermareesei glucoamylase of SEQ ID NO: 230).Paragraph [39]. The recombinant yeast cell of any one of paragraphs[32]-[38], wherein the fermenting organism comprises a heterologouspolynucleotide encoding an alpha-amylase.Paragraph [40]. The recombinant yeast cell of paragraph [39], whereinthe alpha-amylase has a mature polypeptide sequence of at least 60%,e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of any one of SEQ ID NOs:76-101, 121-174 and 231.Paragraph [41]. The recombinant yeast cell of any one of paragraphs[32]-[40], wherein the fermenting organism comprises a heterologouspolynucleotide encoding a protease.Paragraph [42]. The recombinant yeast cell of paragraph [41], whereinthe protease has a mature polypeptide sequence of at least 60%, e.g., atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs: 9-73(e.g., any one of SEQ ID NOs: 9, 14, 16, 21, 22, 33, 41, 45, 61, 62, 66,67, and 69; such as any one of SEQ NOs: 9, 14, 16, and 69).Paragraph [43]. The recombinant yeast cell of any one of paragraphs[32]-[42], wherein the fermenting organism comprises a heterologouspolynucleotide encoding a trehalase.Paragraph [44]. The recombinant yeast cell of paragraph [43], whereinthe trehalase has a mature polypeptide sequence of at least 60%, e.g.,at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of any one of SEQ ID NOs:175-226.Paragraph [45]. The recombinant yeast of any one of paragraphs[32]-[44], wherein the cell is a Saccharomyces, Rhodotorula,Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Rhodosporidium,Candida, Yarrowia, Lipomyces, Cryptococcus, or Dekkera sp. cell.Paragraph [46]. The recombinant yeast of paragraph [45], wherein thecell is a Saccharomyces cerevisiae cell.Paragraph [47]. The recombinant cell of any one of paragraphs [32]-[46],wherein the cell is capable of higher yield of fermentation productand/or reduced foam accumulation when compared to fermentation using thesame process and an identical cell without the heterologouspolynucleotide encoding the phospholipase under the same conditions(e.g., at about or after 54 hours fermentation, such as the conditionsdescribed in Examples 3 or 4).Paragraph [48]. The recombinant cell of paragraph [47], wherein the cellis capable of at least 0.25% (e.g., 0.5%, 0.75%, 1.0%, 1.25%, 1.5%,1.75%, 2%, 3% or 5%) higher yield of fermentation product.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control. Allreferences are specifically incorporated by reference for that which isdescribed.

The following examples are offered to illustrate certain aspects of thepresent invention, but not in any way intended to limit the scope of theinvention as claimed.

EXAMPLES Materials and Methods

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

ETHANOL RED® (“ER”): Saccharomyces cerevisiae yeast available fromFermentis/Lesaffre, USA.

YPD+clonNAT plates were composed of 10 g of yeast extract, 20 g ofpeptone, 20 g bacto agar, and deionized water to 960 ml, followed byautoclave treatment. 40 mL sterile 50% glucose and 1 mL clonNAT stocksolution was added, followed by mixing and pouring.

clonNAT stock solution was composed of 2 g nourseothricin sulfate anddeionized water to 20 ml.

Example 1: Construction of Yeast Strains Expressing a HeterolociousPhospholipase

This example describes the construction of yeast cells containing aheterologous phospholipase under control of a S. cerevisiae TDH3, TEF2,PGK1, ADH1 or RPL18B promoter. Three pieces of DNA containing thepromoter, gene and terminator were designed to allow for homologousrecombination between the three DNA fragments and into the X-3 locus ofthe yeast MHCT-484 (WO2018/222990). The resulting strain has onepromoter-containing fragment (left fragment), one gene-containingfragment (middle fragment) and one PRM9 terminator fragment (rightfragment) integrated into the S. cerevisiae genome at the X-3 locus.

Construction of the Promoter Containing Fragments (Left Fragments)

Synthetic linear uncloned DNA containing 60 bp homology to the X-3 site,S. cerevisiae promoter TDH3, TEF2, PGK1, ADH1 or RPL18B (SEQ ID NOs: 1,2, 4, 5, and 6, respectively) and coding sequence for the S. cerevisiaeMF1α signal peptide (SEQ ID NO: 7) were synthesized by Thermo FisherScientific (Waltham, Mass.). To generate additional linear DNA fortransformation into yeast, each of the five linear DNAs containing theleft cassette above was PCR amplified.

Construction of the Terminator Containing Fragment (Right Fragment)

Synthetic linear uncloned DNA containing the S. cerevisiae PRM9terminator (SEQ ID NO: 243) and 300 bp homology to the X-3 site wassynthetized by Thermo Fisher Scientific.

Construction of the Gene Containing Fragment (Middle Fragment)

Synthetic linear uncloned DNA containing coding sequence for the S.cerevisiae MF1α signal peptide, coding sequence for the maturepolypeptide and 50 bp PRM9 terminator was synthetized by Thermo FisherScientific.

Integration of the Left, Middle and Right-Hand Fragments to GenerateYeast Strains with a Heterologous Phospholipase

The yeast MHCT-484 (WO2018/222990) was transformed with the left, middleand right integration fragments described above. In each transformationpool a fixed left fragment and right fragment were used as well as afixed middle fragment containing the phospholipase gene with 100 ng ofeach fragment. To aid homologous recombination of the left, middle andright fragments at the genomic X-3 sites a plasmid containing Cas9 andguide RNA specific to X-3 (pMcTs442) was also used in thetransformation. These four components were transformed into the S.cerevisiae strain MHCT-484 following a yeast electroporation protocol.Transformants were selected on YPD+cloNAT to select for transformantsthat contain the CRISPR/Cas9 plasmid pMcTs442. Transformants were pickedusing a Q-pix Colony Picking System (Molecular Devices; San Jose,Calif.) to inoculate one well of 96-well plate containing YPD+cloNATmedia. The plates were grown for 2 days then glycerol was added to 20%final concentration and the plates were stored at −80° C. until needed.Integration of specific phospholipase construct was verified by PCR withlocus specific primers and subsequent sequencing. The strains generatedin this example are shown in Table 5.

TABLE 5 SEQ ID NO: Strain (mature Donor Organism Name Promoterpolypeptide) (catalytic domain) HP21-B03 pADH1v1 236 Talaromycesleycettanus HP21-C03 pADH1v1 236 Talaromyces leycettanus HP21-D04 pTEF2236 Talaromyces leycettanus HP21-D05 pTDH3 236 Talaromyces leycettanusHP21-E01 pPGK1 236 Talaromyces leycettanus HP21-F01 pPGK1 236Talaromyces leycettanus HP21-G06 pRPL18B 236 Talaromyces leycettanusHP21-A02 pADH1v1 237 Penicillium emersonii HP21-F04 pTDH3 237Penicillium emersonii HP21-F05 pRPL18B 237 Penicillium emersoniiHP21-A04 pTEF2 240 Kionochaeta sp. HP21-B01 pPGK1 240 Kionochaeta sp.HP21-C05 pTDH3 240 Kionochaeta sp. HP21-G02 pADH1v1 240 Kionochaeta sp.HP21-B05 pTDH3 241 Mariannaea pinicola HP21-B06 pRPL18B 241 Mariannaeapinicola HP21-C06 pRPL18B 241 Mariannaea pinicola HP21-F02 pADH1v1 241Mariannaea pinicola HP21-H03 pTEF2 241 Mariannaea pinicola HP21-G04pTDH3 239 Pseudomonas sp. 62186 HP21-G05 pRPL18B 239 Pseudomonas sp.62186 HP21-H05 pRPL18B 239 Pseudomonas sp. 62186 HP21-A03 pADH1v1 238Bacillus thuringiensis HP21-B04 pTEF2 238 Bacillus thuringiensisHP21-C01 pPGK1 238 Bacillus thuringiensis HP21-C04 pTEF2 238 Bacillusthuringiensis HP21-D01 pPGK1 238 Bacillus thuringiensis HP21-D06 pRPL18B238 Bacillus thuringiensis HP21-E06 pRPL18B 238 Bacillus thuringiensisHP21-F06 pRPL18B 238 Bacillus thuringiensis HP21-H02 pADH1v1 238Bacillus thuringiensis HP21-D03 pADH1v1 235 Thermomyces lanuginosusHP21-E04 pTEF2 235 Thermomyces lanuginosus HP21-E05 pTDH3 235Thermomyces lanuginosus HP21-G01 pPGK1 235 Thermomyces lanuginosusHP21-H06 pRPL18B 235 Thermomyces lanuginosus HP21-A01 pPGK1 242Fictibacillus macauensis HP21-A05 pTDH3 242 Fictibacillus macauensisHP21-A06 pRPL18B 242 Fictibacillus macauensis HP21-F03 pTEF2 242Fictibacillus macauensis HP21-G03 pTEF2 242 Fictibacillus macauensisHP21-H04 pTDH3 242 Fictibacillus macauensis

Example 2: Activity Assay and Small-Scale Fermentation of Yeast StrainsExpressing Phospholipase Preparation of Yeast Cells for Activity Assays

Yeast strains were cultivated for 24 hours in standard YPD mediacontaining 2% glucose. After the cultivation, samples were centrifuged,and the supernatants assayed for enzyme activity as described below.

PLA (Phospholipase A) Activity Assay

PLA activity was detected by using the EnzChek® PLA1 Kit from Invitrogen(Carlsbad, Calif.). The EnzChek® Direct Phospholipase A Assay Kitmeasures phosphatidylcholine-specific phospholipase C (PC-PLC) activity.PLA1 hydrolyzes the ester linkage of phospholipids and fatty acids. TheEnzChek® Phospholipase A1 substrate (PED-A1) is a dye-labeledglycerophosphoethanolamines with BODIPY® FL dye-labeled acyl chain. PLAassay measures the release of the dye at a fluorescence emission at 515nm. Reaction conditions are described in Table 6.

Initial Preparation of Solutions for PLA1 Activity Assay:

-   -   1. Reaction Buffer: 100 mM MOPS+0.5 mM Zn pH 7    -   2. PLA substrate:        -   a. 40 ul DMSO (Comp E) to one vial PLA sub (Comp A)        -   b. Protected from light        -   c. Sufficient volume for 100 rxns    -   3. 500 U/mL stock of positive control PLA (Comp D)        -   a. Dissolved Comp D vial in 100 uL of reaction buffer    -   4. 10 mM DOPC (Comp B)        -   a. Dissolved Comp B vial in 100 ul of ETOH    -   5. 10 mM DOPG (Comp C)        -   a. Dissolved Comp C vial in 100 ul of ETOH

Final Preparation of Solutions and Samples for PLA1 Activity Assay:

-   -   1. 10U/mL positive control made by diluting 500U/mL stock        positive control in reaction buffer        -   a. Example: added 20 ul of 500 U/mL positive control to 980            mL of Reaction buffer    -   2. Serial dilutions of positive control to obtain 8-point        standard curve with initial concentration beginning at 10 U/ml        -   a. Final positive control concentration two-fold lower (5            U/ml) when substrate added    -   3. When necessary, samples diluted in reaction buffer    -   4. Lipid Mix: 30 ul 10 mM DOPC, 30 ul 10 mM DOPG, 30 ul 1 mM PLA        substrate    -   5. PLA substrate        -   a. 50 ul lipid mixed slowly to 5 mL reaction buffer in a            smaller beaker containing a stir bar.        -   b. Stirred for ˜2-5 minutes

Assay Protocol for PLA1 Activity Assay:

-   -   1. 50 ul of 8-point standard curve to columns 10, 11, 12        -   a. last row left blank for buffer    -   2. Added 50 ul of samples to remaining wells    -   3. Added 50 ul of PLA-lipid substrate to wells containing        samples and controls. Mixed well without introducing bubbles.    -   4. Read a T0 at 505EX/515EM (450EX/515EM is ok as well)    -   5. Covered plate and incubate protected from light    -   6. Read after 5 hours of incubation

TABLE 6 PLA1 Activity Assay Condition Amount of yeast supernatant 50 μlAmount of PLA-lipid substrate 50 μl Substrate PLA1 substrate from KitBuffer 100 mM MOPS + 0.5 mM Zn pH 7.0 ± 0.05 Incubation temperature 22°C. (room Temperature) Reaction time 5 hrs Wavelength 505EX/515EM

PLC (Phospholipase C) Activity Assay

PLC activity was detected by using EnzChek® PLC Kit from Invitrogen. TheEnzChek® Phospholipase A Assay Kit measures phosphatidylcholine-specificphospholipase C (PC-PLC) activity by measuring the amount of starchdegraded through enzymatic hydrolysis of starch. The assay uses aproprietary substrate (glycero-phosphoethanolamine with a dye-labeledsn-2 acyl chain) to detect PLC activity. Substrate cleavage by PLCreleases the dye-labeled diacylglycerol, which produces a fluorescencesignal that can be measure at 516 nm emission. Reaction conditions aredescribed in Table 7.

Initial Preparation of Solutions for PLC Activity Assay:

-   -   1. Reaction Buffer: 100 mM NaAc+0.5 mM Zn pH 5    -   2. 200× stock of PLC substrate        -   a. 100 ul DMSO (Comp B) added to one vial PLC sub        -   b. Protected from light        -   c. Sufficient volume for 125 rxns    -   3. 40 U/mL stock of positive control PC-PLC (Comp E)        -   a. Dissolved Comp E vial in 200 uL of reaction buffer

Final Secondary Preparation of Solutions and Samples for PLC ActivityAssay

-   -   1. 1U/mL positive control made by diluting 40U/mL stock positive        control 40-fold a. Example: added 25 ul of 40 U/mL positive        control to 0.975 mL of Reaction buffer    -   2. Serial dilutions of positive control to obtain 8-point        standard curve with initial concentration beginning at 0.125        U/ml        -   a. Final positive control concentration two-fold lower            (0.0625 U/ml) when substrate is added    -   3. When necessary, samples diluted in reaction buffer    -   4. PLC substrate: Added 40 ul of lecithin (Comp D) and 100 ul of        PLC Substrate 200× stock solution (prepared in earlier step) to        9.86 mL of reaction buffer.

Assay Protocol for PLC Activity Assay:

-   -   1. 75 ul of 8-point standard curve to columns 10, 11, 12        -   a. last row left blank for buffer    -   2. Added 75 ul of samples to remaining wells    -   3. Added 75 ul of PLC substrate to wells containing samples and        controls. Mixed well without introducing bubbles.    -   4. Read a T0 at 509EX/516EM (490EX/520EM is ok as well)    -   5. Covered plate and incubate protected from light    -   6. Read after 5 hours

TABLE 7 PLC Activity Assay Condition Amount of yeast supernatant 75 μlAmount of PLA-lipid substrate 75 μl Substrate PLC substrate from KitBuffer 100 mM NaAc + 0.5 mM Zn pH 5.0 ± 0.05 Incubation temperature 22°C. (room Temperature) Reaction time 5 hrs Wavelength 509EX/516EM

Preparation of Yeast Culture for Microtiter Plate Fermentations

Simultaneous saccharification and fermentation (SSF) was performed viamini-scale fermentations using industrial corn mash (Avantec® Amp;Novozymes A/S). Yeast strains were cultivated overnight in YPD mediawith 6% glucose for 24 hours at 30° C. and 300 rpm. The corn mash wassupplemented with 250 ppm of urea and dosed with 0.45 AGU/g-DS of anexogenous glucoamylase enzyme product (Spirizyme® Excel; Novozymes A/S).Approximately 0.6 mL of corn mash was dispensed per well to 96 wellmicrotiter plates, followed by the addition of approximately10{circumflex over ( )}8 yeast cells/g of corn mash from the overnightculture. Plates were incubated at 32° C. without shaking. Fermentationwas stopped by the addition of 100 μL of 8% H₂SO₄, followed bycentrifugation at 3000 rpm for 10 min. The supernatant was analyzed forethanol using HPLC. Fermentation reaction conditions are summarized inTable 8. SSF and activity assay results of yeast strains expressing aphospholipase are in Table 9.

TABLE 8 Microtiter plate fermentation reaction conditions SubstrateAvantec ® Amp corn mash Yeast pitch 10{circumflex over ( )}8 cells/gcorn mash Supplementary urea 250 ppm Exogenous glucoamylase product dose0.15 AGU/g-DS pH 5.0 ± 0.05 Incubation temperature 32° C. Reaction time48 hours

TABLE 9 Phospholipase yeast strains enzyme activity measurements andethanol titers from SSF SEQ ID NO: PLC PLA1 Mean (mature activityactivity Ethanol Strain ID Promoter polypeptide) units units (g/L)MHCT-484 none none 99 0 107.31 HP21-D04 TEF2 236 776 23 110.67 HP21-G06RPL18B 236 793 0 110.6105 HP21-B03 ADH1v1 236 863 43 110.2833 HP21-D05TDH3 236 688 29 110.0453 HP21-C03 ADH1v1 236 878 47 108.885 HP21-E01PGK1 236 710 11 108.2008 HP21-F01 PGK1 236 680 0 106.1183 HP21-F04 TDH3237 207 0 110.67 HP21-F05 RPL18B 237 174 0 109.1528 HP21-A02 ADH1v1 237273 0 109.0933 HP21-B01 PGK1 240 452 0 94.0695 HP21-A04 TEF2 240 451 083.27025 HP21-G02 ADH1v1 240 410 0 82.19925 HP21-C05 TDH3 240 426 081.36625 HP21-C06 RPL18B 241 236 0 112.455 HP21-B06 RPL18B 241 201 0107.8735 HP21-B05 TDH3 241 356 0 100.436 HP21-H03 TEF2 241 275 0 99.3055HP21-F02 ADH1v1 241 364 0 97.58 HP21-G04 TDH3 239 85 0 107.1893 HP21-G05RPL18B 239 251 0 84.7875 HP21-H05 RPL18B 239 220 0 84.609 HP21-E06RPL18B 238 156 0 109.599 HP21-B04 TEF2 238 152 0 108.6768 HP21-D01 PGK1238 99 0 108.0223 HP21-C01 PGK1 238 116 0 107.5463 HP21-F06 RPL18B 238142 0 105.077 HP21-A03 ADH1v1 238 99 0 104.5118 HP21-H02 ADH1v1 238 93 0102.102 HP21-C04 TEF2 238 146 0 101.9235 HP21-D06 RPL18B 238 141 082.25875 HP21-E05 TDH3 235 846 31 110.5808 HP21-H06 RPL18B 235 685 56109.0635 HP21-E04 TEF2 235 500 139 100.5253 HP21-G01 PGK1 235 428 12396.06275 HP21-D03 ADH1v1 235 462 142 88.08975 HP21-F03 TEF2 242 115 0108.171 HP21-A05 TDH3 242 126 0 108.0818 HP21-A06 RPL18B 242 135 0106.2373 HP21-G03 TEF2 242 113 0 105.791 HP21-H04 TDH3 242 102 0104.6903 HP21-A01 PGK1 242 91 0 104.4523

Example 3: Fermentation of Yeast Strains Expressing Phospholipase

The purpose of this experiment was to determine if expressingphospholipase in yeast would enhance ethanol yields at the end offermentation. Yeast strains (HP21-H06, HP21-D05, HP21-G12, HP21-A08,HP21-F05 and HP21-F04) were propagated overnight, and then used to dosea fermentation of industrially liquified mash. The fermented mash wassampled at the end of fermentation and the supernatant was filtered andanalyzed by HPLC to determine the final ethanol titers and residualsugars.

Yeast Propagation

Yeast strains were received as glycerol stocks and were kept frozen at−80° C. until used. The yeast strains were propagated according to thefollowing procedure:

-   -   1. Cryo-vials of yeast were removed from the −80° C. freezer.    -   2. For each sample, 2×50 mL sterile Erlenmeyer flasks were        obtained and labeled.    -   3. The flasks were placed into a sterile hood along with 6% YPD        media, pipettes and tips, and cryovials.    -   4. Using sterile technique, ˜50 mL of 6% YPD media was poured        into each bottle.    -   5. 150 uL of the appropriate yeast sample was added to each        flask using a sterile 200 uL pipette and sterile tips.    -   6. The flasks were capped after each addition to maintain a        sterile solution.    -   7. The flasks were placed into a 32° C. shaking incubator and        mixed at ˜150 rpm overnight.

Cell Counting

The propagation slurries described above were then tested to determinethe number of yeast cells present for dosing, according to the followingprocedure:

-   -   1. The propagated yeast samples were removed from the incubator.    -   2. The 50 mL samples were poured into 50 mL flip top falcon        tubes.    -   3. The samples were centrifuged 3,500 rpm for 7 min.    -   4. The supernatant was discarded into the old 50 mL flasks to be        autoclaved.    -   5. 10 mL of deionized water was added to each tube.    -   6. The tubes were vortex mixed to suspend the pellet.    -   7. The replicate samples were combined into a single 50 mL        falcon tube.    -   8. The samples were centrifuged 3,500 rpm for 7 min.    -   9. The supernatant was discarded into the original 50 mL flasks        to be autoclaved.    -   10. 10 mL of deionized water was added to each remaining tube.    -   11. The tubes were vortex mixed to suspend the pellet.        -   a. This slurry was saved and will be used to dose into the            fermentations.    -   12. The yeast slurries were diluted 100× into 15 mL falcon tubes        by adding 100 uL to 9.9 mL of deionized water.    -   13. The samples subjected to cell count using a NucleoCounter®        YC-100 (Chemometec A/S) as follows:        -   a. 50 uL of the 100× slurry dilution was added to 450 uL of            Lysis buffer.        -   b. The samples were vortexed and allowed to sit ˜5 min for            the cell lysis to occur.        -   c. A nucleo-casette was used to sample the lysed solution,            then placed into the NucleoCounter® YC-100.        -   d. The NucleoCounter® YC-100 analyzed the slurry and            produced a cell count number.        -   e. The resulting number is multiplied by 1000 to get the            final cell counts for use in the spreadsheet.    -   14. The yeasts used in this experiment, and the yeast counts        that were obtained according to the procedure above are        displayed in the Table 10 below.

TABLE 10 Average Strain ID YCL-1 YCL-2 total cells MHCT-484 1.26E+091.28E+09 1.27E+09 HP21-H06 1.23E+09 1.16E+09 1.19E+09 HP21-D05 1.38E+091.43E+09 1.40E+09 HP21-G12 1.14E+09 1.12E+09 1.13E+09 HP21-A08 1.38E+091.29E+09 1.33E+09 HP21-F05 1.28E+09 1.28E+09 1.28E+09 HP21-F04 1.41E+091.24E+09 1.33E+09

-   -   15. The calculation below was used to determine the number of mL        of yeast slurry to add to each tube.

${\frac{\text{10,000,000 cells}}{{mL}\mspace{14mu}{mash}} \times \frac{1\mspace{14mu}{mL}\mspace{14mu}{mash}}{1.15\mspace{14mu} g\mspace{14mu}{mash}} \times g\mspace{14mu}{mash} \times \frac{mL}{X\mspace{14mu}{cells}} \times \frac{1000\mspace{14mu}\mu\; L}{mL}} = {\mu\; L\mspace{14mu}{yeast}\mspace{11mu}{to}\mspace{14mu}{add}}$

Fermentation

Fermentations of industrially liquified mash were conducted according tothe following procedure:

-   -   1. Industrially liquefied mash was acquired and stored frozen        for future analysis. The mash used in this experiment was from a        plant using Avantec® Amp and running a hydro-heater or jet        cooker during liquefaction stage.    -   2. Two liters of the mash was thawed for approximately 2 hours        prior to starting this study.    -   3. The mash was adjusted to 500 ppm urea and 3 ppm penicillin        using stock solutions of 200 g/L urea and 1 g/L penicillin.    -   4. The mash was adjusted to pH 5 using 40% v/v H₂SO₄ and the dry        solids content were measured on a Mettler-Toledo moisture        balance.    -   5. 50.0+/−0.05 g of prepared mash was weighed into 250 mL media        bottles.    -   6. The bottles were capped and stored in a refrigerator        overnight.    -   7. The bottles were dosed with enzymes and yeast in the morning        using a repeater pipette.    -   8. The conditions tested include Achieve® glucoamylase (0.42        AGU/gDS), trehalase (2 μg/gDS), and yeast dosed to approximately        7,000,000 cells per fermenation.    -   9. The bottles were capped with hole drilled caps. The holes        were covered with labeling tape. A 0.24-gauge syringe needle was        used to pierce a uniform vent hole to allow for CO₂ gas to        escape during fermentation.    -   10. The bottles were placed into a 32° C. air shaker set to 150        rpm to begin fermentation.    -   11. Fermentation was run for 54 hours.

Sampling of Fermentations

-   -   1. After 52 hours of fermentation, 5 g of fermented mash were        pipetted into a 15 mL conical falcon tube with 5 mL pipette        equipped with a cut tip.    -   2. 50 uL of 40% H₂SO₄ were added to each of the tubes to stop        fermentation and enzymatic hydrolysis.    -   3. The tubes were briefly mixed with a vortex mixer and        centrifuged at 3,500 rpm for 7 minutes.    -   4. The supernatant was poured into a syringe equipped with a        0.45 um filter and the plunger of the syringe was used to push        the sample through the filter into prenumbered HPLC vials.    -   5. The HPLC vials were capped and the samples were submitted to        HPLC for analysis of the ethanol and sugars produced during        fermentation. The HPLC setup was as shown in Table 11.

TABLE 11 HPLC system Agilent's 1100/1200 series with Chem stationsoftware Degasser Quaternary Pump Auto-Sampler Column Compartment/wHeater Refractive Index Detector (RI) Column Bio-Rad HPX- 87H IonExclusion Column 300 mm × 7.8 mm parts# 125-0140 Bio-Rad guard cartridgecation H parts# 125- 0129, Holder parts# 125-0131 Method 0.005M H₂SO₄mobile phase Flow rate of 0.6 ml/min Column temperature - 65° C. RIdetector temperature - 55° C.

The method quantifies analytes using calibration standards for dextrins(DP4+), maltotriose, maltose, glucose, fructose, acetic acid, lacticacid, glycerol and ethanol. A 4-point calibration including the originwas used.

Ethanol improvement for tested strains is shown in FIG. 1. StrainsHP21-F05 and HP21-F04 show approximately 1.1% improvement in ethanolover control strain MHCT-484, while strain HP21-A08 showed approximately0.5% improvement in ethanol titer of the control.

Example 4: Fermentation of Yeast Strains Expressing Phospholipase atVarying Urea Levels

The purpose of this experiment was to examine the effect of nitrogenloading (urea level) on ethanol yields of phospholipase-expressingyeasts. Yeast strains (HP21-H08, HP21-F04 and HP21-F05) were propagatedovernight, and then used to dose a fermentation of industriallyliquified mash. The fermented mash was sampled at the end offermentation and the supernatant was filtered and run on HPLC todetermine the final ethanol titers and residual sugars.

Yeast Propagation

Yeast strains were propagated as described above in Example 3.

Cell Counting

The propagation slurries described above were then tested to determinethe number of yeast cells present for dosing according to the proceduredescribed above in Example 3. The yeasts used in this experiment, andthe corresponding yeast counts are displayed in the Table 12.

TABLE 12 Average Strain ID YCL-1 YCL-2 total cells MHCT-484 1.48E+091.44E+09 1.46E+09 HP21-A08 1.24E+09 1.24E+09 1.24E+09 HP21-F05 1.50E+091.48E+09 1.49E+09 HP21-F04 1.37E+09 1.40E+09 1.38E+09

Fermentation

Fermentations of industrially liquified mash were conducted according tothe following procedure:

-   -   1. Industrially liquefied mash was acquired and stored frozen        for future analysis. The two mashes used in this experiment were        from a plant using Avantec® Amp (Novozymes A/S) and a plant        running another product Liquozyme® Pro (Novozymes A/S). Both        mashes were from plants running a hydro-heater or jet cooker        during liquefaction stage.    -   2. Two liters of each mash was thawed for approximately 2 hours        prior to starting this study.    -   3. The mash was dosed with 3 ppm penicillin using a stock        solution 1 g/L penicillin. Urea was added to the fermentation        during enzyme dosing using stock solutions of 100 and 20 g/L.    -   4. The mash was adjusted to pH 5 using 40% v/v H₂SO₄ and the dry        solids content were measured on a Mettler-Toledo moisture        balance.    -   5. ˜5 g of the prepared mash was added to preweighed 15 mL tubes    -   6. The tubes were reweighed and the weight of mash was used in        calculating enzymes, yeast and urea dosing.    -   7. The tubes were capped and stored in a refrigerator overnight.    -   8. The tubes were dosed with enzymes, yeast and urea in the        morning using a Biomek FX liquid handling robot.    -   9. The conditions tested in this experiment are displayed in        Table 13 below. Yeast dose in this experiment was 7,000,000        cells per ferm.    -   10. The tubes were capped with holes to allow released CO₂ from        fermentation.    -   11. Tubes were placed into a 32° C. incubator to begin        fermentation.    -   12. Tubes were mixed with a vortex mixer twice a day for 52        hours.

TABLE 13 Treatment # Yeast# Glucoamylase Dose Units Trehalase Dose UnitsPLC Dose Units Urea Dose Units MHCT-484 - 1 1 Achieve 0.42 AGU/gDSTrehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 0 ppm Control MHCT-484 -2 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 5 μg/g DS Urea0 ppm 5 ug PLC MHCT-484 - 3 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P.emersonii 20 μg/g DS Urea 0 ppm 20 ug PLC HP21-A08 4 2 Achieve 0.42AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 0 ppm HP21-F05 53 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 0ppm HP21-F04 6 4 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0μg/g DS Urea 0 ppm MHCT-484 - 7 1 Achieve 0.42 AGU/gDS Trehelase 1ug/gDS P. emersonii 0 μg/g DS Urea 150 ppm Control MHCT-484 - 8 1Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 5 μg/g DS Urea 150ppm 5 ug PLC MHCT-484 - 9 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P.emersonii 20 μg/g DS Urea 150 ppm 20 ug PLC HP21-A08 10 2 Achieve 0.42AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 150 ppm HP21-F0511 3 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea150 ppm HP21-F04 12 4 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P.emersonii 0 μg/g DS Urea 150 ppm MHCT-484 - 13 1 Achieve 0.42 AGU/gDSTrehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 500 ppm ControlMHCT-484 - 14 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 5μg/g DS Urea 500 ppm 5 ug PLC MHCT-484 - 15 1 Achieve 0.42 AGU/gDSTrehelase 1 ug/gDS P. emersonii 20 μg/g DS Urea 500 ppm 20 ug PLCHP21-A08 16 2 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0μg/g DS Urea 500 ppm HP21-F05 17 3 Achieve 0.42 AGU/gDS Trehelase 1ug/gDS P. emersonii 0 μg/g DS Urea 500 ppm HP21-F04 18 4 Achieve 0.42AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 500 ppmMHCT-484 - 19 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0μg/g DS Urea 0 ppm Control MHCT-484 - 20 1 Achieve 0.42 AGU/gDSTrehelase 1 ug/gDS P. emersonii 5 μg/g DS Urea 0 ppm 5 ug PLC MHCT-484 -21 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 20 μg/g DSUrea 0 ppm 20 ug PLC HP21-A08 22 2 Achieve 0.42 AGU/gDS Trehelase 1ug/gDS P. emersonii 0 μg/g DS Urea 0 ppm HP21-F05 23 3 Achieve 0.42AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 0 ppm HP21-F04 244 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 0ppm MHCT-484 - 25 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii0 μg/g DS Urea 300 ppm Control MHCT-484 - 26 1 Achieve 0.42 AGU/gDSTrehelase 1 ug/gDS P. emersonii 5 μg/g DS Urea 300 ppm 5 ug PLCMHCT-484 - 27 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 20μg/g DS Urea 300 ppm 20 ug PLC HP21-A08 28 2 Achieve 0.42 AGU/gDSTrehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 300 ppm HP21-F05 29 3Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 300ppm HP21-F04 30 4 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0μg/g DS Urea 300 ppm MHCT-484 - 31 1 Achieve 0.42 AGU/gDS Trehelase 1ug/gDS P. emersonii 0 μg/g DS Urea 1000 ppm Control MHCT-484 - 32 1Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 5 μg/g DS Urea 1000ppm 5 ug PLC MHCT-484 - 33 1 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P.emersonii 20 μg/g DS Urea 1000 ppm 20 ug PLC HP21-A08 34 2 Achieve 0.42AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea 1000 ppm HP21-F0535 3 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P. emersonii 0 μg/g DS Urea1000 ppm HP21-F04 36 4 Achieve 0.42 AGU/gDS Trehelase 1 ug/gDS P.emersonii 0 μg/g DS Urea 1000 ppm

Sampling of Fermentations

-   -   1. After 52 hours of fermentation, 50 uL of 40% H₂SO₄ were added        to each of the tubes to stop fermentation and enzymatic        hydrolysis.    -   2. The tubes were briefly mixed with a vortex mixer and        centrifuged at 3,500 rpm for 7 minutes.    -   3. The tubes were placed onto a Biomek liquid handler which was        used to pipette 200 uL of supernatant from the tubes into a 96        well 0.22 um filter plate.    -   4. The filter plate was placed on top of a round bottom 96 well        polypropylene plate.    -   5. The filter and plate were placed into a floor centrifuge with        bioseal caps on the buckets and spun at 3,500 rpm for 7 minutes        or until liquid had passed through all the wells of the 96 well        filter plate.    -   6. The plates were removed from the centrifuge and the round        bottom plate with the filtered sample was heat sealed and        submitted to HPLC for analysis of the ethanol and sugars        produced during fermentation. The HPLC setup was as shown in        Table 14.

TABLE 14 HPLC system Agilent's 1100/1200 series with Chem stationsoftware Degasser Quaternary Pump Auto-Sampler Column Compartment/wHeater Refractive Index Detector (RI) Column Bio-Rad HPX- 87H IonExclusion Column 300 mm × 7.8 mm parts# 125-0140 Bio-Rad guard cartridgecation H parts# 125- 0129, Holder parts# 125-0131 Method 0.005M H₂SO₄mobile phase Flow rate of 0.6 ml/min Column temperature - 65° C. RIdetector temperature - 55° C.

The method quantifies analytes using calibration standards for dextrins(DP4+), maltotriose, maltose, glucose, fructose, acetic acid, lacticacid, glycerol and ethanol. A 4-point calibration including the originwas used. Normalized mean ethanol values after 54 hours of fermentationof AMP mash at 0 and 150 ppm urea are shown in FIG. 2.

All tested strains expressing phospholipase showed greater increases inethanol titer with no urea loading. Normalized mean ethanol values after54 hours of fermentation of non-AMP mash at 0 and 150 ppm urea are shownin FIG. 3. All tested strains expressing phospholipase showed greaterincreases in ethanol titer with no urea loading.

Example 5: Construction of Additional Yeast Strains Expressing aHeterologous Phospholipase

This example describes the construction of yeast cells containing theremaining heterologous phospholipases of Table 1 under the control of anS. cerevisiae PGK1 promoter. Three pieces of DNA containing thepromoter, gene and terminator were designed to allow for homologousrecombination between the four DNA fragments and into the XII-2 locus ofthe strain MeJi797 (a derivative of MBG5012 expressing bothalpha-amylase and glucoamylase; WO2019/161227). The resulting strain hasone promoter-containing fragment (left fragment), one gene-containingfragment (middle fragment) and one PRM9 terminator fragment (rightfragment) integrated into the S. cerevisiae genome at the XII-2 locus.

Construction of the Promoter-Containing Fragment (Left Fragment)

Linear DNA containing 500 bp homology to the XII-2 site and the S.cerevisiae pPGK1 promoter was PCR amplified from HP27 plasmid DNA withprimers 1229945 (5′-TCTTT TCGCG CCCTG GAAAG G-3′; SEQ ID NO: 434) and1227122 (5′-TGTTT TATAT TTGTT GTAAA AAGTA GATAA TTACT TCCTT GATGATCTG-3′; SEQ ID NO: 435). Fifty pmoles each of forward and reverseprimer was used in a PCR reaction containing 5 ng of plasmid DNA astemplate, 0.1 mM each dATP, dGTP, dCTP, dTTP, 1× Phusion HF Buffer(Thermo Fisher Scienctific), and 2 units Phusion Hot Start DNApolymerase in a final volume of 50 μL. The PCR was performed in a T100™Thermal Cycler (Bio-Rad Laboratories, Inc.) programmed for one cycle at98° C. for 30 seconds followed by 32 cycles each at 98° C. for 10seconds, 59° C. for 20 seconds, and 72° C. for 40 seconds with a finalextension at 72° C. for 10 minutes. Following thermocycling, the PCRreaction products gel isolated and cleaned up using the NucleoSpin Geland PCR clean-up kit (Machery-Nagel).

The linear DNA containing 243 bp homology to the S. cerevisiae pPGK1promoter (SEQ ID NO: 4) and the MF1α signal peptide (SEQ ID NO: 7) wasPCR amplified from DNA synthesized by GeneArt with primers 1229946(5′-GTGAC AACAA CAGCC TGTTC TC-3′; SEQ ID NO: 436) and 1222995 (5′-AGCTAATGCG GAGGA TGCTG C-3′; SEQ ID NO: 437). Fifty pmoles each of forwardand reverse primer was used in a PCR reaction containing 5 ng of plasmidDNA as template, 0.1 mM each dATP, dGTP, dCTP, dTTP, 1× Phusion HFBuffer (Thermo Fisher Scientific), and 2 units Phusion Hot Start DNApolymerase in a final volume of 50 μL. The PCR was performed in a T100™Thermal Cycler (Bio-Rad Laboratories, Inc.) programmed for one cycle at98° C. for 30 seconds followed by 32 cycles each at 98° C. for 10seconds, 59° C. for 20 seconds, and 72° C. for 20 seconds with a finalextension at 72° C. for 5 minutes. Following thermocycling, the PCRreaction products gel isolated and cleaned up using the NucleoSpin Geland PCR clean-up kit (Machery-Nagel).

Construction of the Terminator-Containing Fragment (Right Fragment)

The DNA containing 250 bp of the PRM9 terminator and 500 bp of the XII-23′-end homology was PCR amplified from TH12 plasmid DNA (GeneArt) withprimers 1221473 (5′-ACAGA AGACG GGAGA CACTA GC-3′; SEQ ID NO: 438) and1229949 (5′-GGGGT CGCAA CTTTT CCC-3′; SEQ ID NO: 439). Fifty pmoles eachof forward and reverse primer was used in a PCR reaction containing 5 ngof plasmid DNA as template, 0.1 mM each dATP, dGTP, dCTP, dTTP, lxPhusion HF Buffer (Thermo Fisher Scientific), and 2 units Phusion HotStart DNA polymerase in a final volume of 50 μL. The PCR was performedin a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.) programmed forone cycle at 98° C. for 30 seconds followed by 32 cycles each at 98° C.for 10 seconds, 59° C. for 20 seconds, and 72° C. for 40 seconds with afinal extension at 72° C. for 10 minutes. Following thermocycling, thePCR reaction products gel isolated and cleaned up using the NucleoSpinGel and PCR clean-up kit (Machery-Nagel).

Construction of the Phospholipase-Containing Fragments (MiddleFragments)

Synthetic linear uncloned DNA containing the MF1α signal peptide,phospholipase gene and 50 bp of the PRM9 terminator were obtained fromGeneart or Twist Bioscience.

Integration of the Left, Middle and Right-Hand Fragments to GenerateYeast Strains with a Heterologous Phosphoplipase

The yeast MeJi797 was transformed with the left, middle and rightintegration fragments described above. In each transformation pool afixed left fragment and right fragment with 100 ng of each fragment wasused. The middle fragment consisted of a signal peptide andphospholipase gene with ˜100 ng of each fragment (700 ng total). To aidhomologous recombination of the left, middle and right fragments at thegenomic XII-2 sites a plasmid containing MAD7 and guide RNA specific toXII-2 (pMIBa638) was also used in the transformation. These fourcomponents were transformed into the into S. cerevisiae strain MeJi797following a yeast electroporation protocol. Transformants were selectedon YPD+cloNAT to select for transformants that contain the Mad7 plasmidpMIBa638. Transformants were picked using a Q-pix Colony Picking System(Molecular Devices) to inoculate one well of 96-well plate containingYPD+cloNAT media. The plates were grown for two days then glycerol wasadded to 20% final concentration and the plates were stored at −80° C.until needed. Integration of specific phospholipase construct wasverified by PCR with locus specific primers and subsequent sequencing.The strains generated were used in the following examples.

Example 6: Corn Mash Fermentations of Phospholipase-Expressing YeastStrains

The strains described in Example 5 were tested for ethanol productionand residual glucose using a 96-well corn mash fermentation describedbelow. Propagation plates were prepared by inoculating 10 μL of eachstrain into a 96-well seed plate containing 150 uL YP+2% glucose mediumper well. Plates were incubated at 30° C. and 300 RPM overnight. Thefollowing day, 10 μL of the seed culture was transferred to 96-deep wellplates containing 500 μL of East Kansas Agri-Energy Liquefact Amp cornmash supplemented with 100 ppm urea and 0.42 AGU/g Spirizyme Excel.Plates were sealed with EnzyScreen plate covers and tightly clamped tolimit oxygen transfer. Corn mash plates were incubated statically at 32°C. for 56 hours. After fermentation was completed, the plates wereplaced at −80 C for about 10 minutes before 100 μL of 8% H₂SO₄ was addedto each well of the 96-deep well corn mash plates. Plates were sealedand mixed by inversion and centrifuged at 3000 rpm for 10 minutes.Supernatants were removed and diluted to 12× in sterile deionized waterprior to HPLC analysis. The average ethanol and residual glucose perintegrated heterologous phospholipase gene are shown in Table 15.

TABLE 15 SEQ ID NO: SEQ (mature mean mean Donor Organism ID NO: poly-ethanol glucose (catalytic domain) (coding) peptide) (g/L) (g/L) N/A(MeJi797 control strain) — — 128.84 4.82 Penicillium cinnamopurpureum423 332 138.52 3.30 Talaromyces rugulosus 407 316 138.39 1.28Aspergillus turcosus 384 293 136.06 1.42 Aspergillus egyptiacus 376 285135.98 2.97 Bacillus mycoides 381 290 135.88 5.16 Penicillium swiecickii392 301 134.40 5.30 Lysinibacillus xylanilyticus 388 297 134.23 1.94Listeria seeligeri 391 300 133.80 3.99 Penicillium spikei 410 319 133.371.68 Talaromyces leycettanus 402 311 133.29 1.02 Bacillus toyonensis 404313 133.10 2.35 Bacillus mycoides 373 282 132.84 5.32 Bacillus mycoides397 306 132.72 5.88 Rasamsonia argillacea 356 265 132.72 0.96 Bacillustoyonensis 427 336 132.66 4.17 Rasamsonia byssochlamydoides 348 257132.53 1.34 Bacillus mycoides 414 323 132.42 3.18 Listeria innocua 375284 132.36 5.22 PeniciIlium simplicissimum 430 339 132.26 2.57 Bacillusmycoides 380 289 132.26 2.40 Talaromyces boninensis 393 302 132.05 1.08Hamigera paravellanea 401 310 131.88 2.04 Penicillium vasconiae 416 325131.76 1.08 Rasamsonia brevistipitata 352 261 131.76 2.52 Bacillusmycoides 422 331 131.68 4.36 Bacillus pseudomycoides 372 281 131.66 3.57Bacillus sp. 428 337 131.60 2.88 Bacillus acidiceler 419 328 131.50 4.78Bacillus manliponensis 429 338 131.36 2.44 Hamigera avellanea 409 318131.23 1.49 Penicillium flavescens 358 267 131.22 4.68 Bacillus sp. 420329 130.98 5.96 Talaromyces bacillisporus 367 276 130.86 3.80Talaromyces cellulolyticus 425 334 130.74 0.84 Rasamsonia eburnea 349258 130.61 2.07 Bacillus acidiceler 433 342 130.52 5.04 Talaromycesrugulosus 364 273 130.52 1.00 Bacillus thuringiensis 396 305 130.46 3.89Brevibacillus sp. 415 324 130.44 18.72 Bacillus drentensis 383 292130.41 6.75 Talaromyces verruculosus 424 333 129.78 3.18 Penicilliumpiscarium 366 275 129.74 4.37 Penicillium sclerotiorum 347 256 129.729.06 Hamigera terricola 365 274 129.68 10.32 Penicillium arenicola 431340 129.65 1.34 Aspergillus tamarii 377 286 128.86 1.03 Penicillium sp.408 317 128.04 1.92 Bacillus sp. 382 291 127.84 3.64 Aspergillustubingensis 386 295 127.66 3.89 Penicillium emersonii 355 264 127.653.75 Talaromyces subinflatus 385 294 127.26 6.60 Bacillus thuringiensis405 314 127.08 3.00 Penicillium vasconiae 361 270 126.96 12.44Talaromyces columbinus 362 271 126.69 4.05 Bacillus bingmayongensis 413322 126.67 6.86 Bacillus luciferensis 379 288 126.51 5.46 Hamigerastriata 394 303 126.36 2.04 Bacillus thuringiensis 406 315 126.30 9.00Bacillus toyonensis 389 298 126.30 11.82 Bacillus mycoides 412 321126.15 13.95 Bacillus acidiceler 387 296 125.85 5.37 Bacillus wiedmannii390 299 125.67 7.25 Talaromyces variabilis 363 272 125.33 5.78 Bacillusthuringiensis 374 283 125.32 4.08 Penicillium simplicissimum 360 269125.04 10.32 Aspergillus stramenius 371 280 124.92 3.52 Penicilliumbrefeldianum 350 259 124.86 17.10 Penicillium bialowiezense 346 255124.72 3.00 Penicillium scabrosum 353 262 124.56 14.67 Penicilliummegasporum 369 278 124.44 2.10 Penicillium donkii 400 309 123.96 0.84Penicillium jensenii 370 279 123.75 5.58 Galactomyces candidus 368 277123.36 14.37 Bacillus sp. 395 304 123.18 8.82 Aspergillus niger 378 287123.00 8.28 Bacillus pseudomycoides 421 330 122.22 4.86 Penicilliumhispanicum 359 268 118.14 19.95 Penicillium manginii 354 263 117.9613.15 Penicillium meridianum 345 254 117.72 14.92 Paenibacillus sp. 403312 110.07 38.28 Paenibacillus alginolyticus 411 320 106.52 43.96

Example 7: Enhancement of Ethanol Yield with Phospholipase-ExpressingYeast Strains

Commercial Amp corn mash was obtained from Trenton Agri at 35.9% (w/w)of dry solids content and was diluted with tape water to 32.0% (w/w).After dilution, the pH value of the mash was adjusted to 5.1 with 39%(w/v) NaOH solution. Urea and lactrol were added into the pH adjustedAmp mash to final concentration at 150 ppm and 3 ppm, respectively. Theprepared corn mash was aliquoted into 250 mL flasks (100 g/flask).

To propagate yeast, 50 mL of 6% YPD and 100 μl of yeast-glycerol stocksolution were mixed in a 125-mL flask and then incubated at 32° C. forovernight. After incubation, 45 mL of propagation was transferred to a50 mL centrifuge tube and centrifuged at 3500 rpm for 10 minutes. Theliquid fraction was decanted and deionized water was used to twice washthe cells. The cells were resuspended in 10 mL of deionized water andthe total and dead cell accounts were measured using a NucleoCounter®YC-100.

The exogenous α-glucoamylase (Spirizyme Achieve-T™; Novozymes A/S) wasadded into the flasks containing corn mash per DOE and mixed well. Thenthe pre-determined amount of yeast suspension was added and mixed well.The fermentation was performed at 32° C. for 54 hours. After thefermentation, 5 g of slurry was taken out and the fermentation wasstopped by adding 50 μL of 40% H₂SO₄. The liquid was separated fromsolid by centrifuging whole slurry at 900 g for 10 min. The ethanolconcentration was measured using HPLC.

Compared with the control strain MHCT-484, both phospholipase-expressingyeast strains showed significantly increased final ethanol yield (13.30%for control strain MHCT-484, to 13.43% and to 13.42% for strainsHP21-F04 and HP21-F05, respectively.

Example 8: Enhancement of Oil Extraction with Phospholipase-ExpressingYeast Strains

Commercial Amp corn mash was obtained from Trenton Agri at 35.9% (w/w)of dry solids content and was diluted water to 32.0% (w/w). Afterdilution, the pH value of the mash was adjusted to 5.1 with 39% (w/v)NaOH solution. Urea and lactrol were added into the pH adjusted Amp mashto final concentration at 150 ppm and 3 ppm, respectively. The preparedcorn mash was aliquoted into 250-mL flasks (100 g/flask).

To propagate yeast, 50 mL of 6% YPD and 100 μL of yeast-glycerol stocksolution were mixed in a 125-mL flask and then incubated at 32° C. forovernight. After incubation, 45 mL of propagation was transferred to a50-mL centrifuge tube and centrifuged at 3500 rpm for 10 minutes. Theliquid fraction was decanted, and deionized water was used to twice washthe cells. The cells were resuspended in 10 mL of deionized water andthe total and dead cell accounts were measured using a NucleoCounter®YC-100.

Exogenous α-glucoamylase (Spirizyme Achieve-T™; Novozymes A/S), wasadded into the flasks containing corn mash per DOE and mixed well. Thenthe pre-determined amount of yeast suspension was added and mixed well.The fermentation was performed at 32° C. for 54 hours. Afterfermentation, 10 mL of 95% hexane was added to 95 grams of whole slurry.The slurry-hexane mixture was mixed well and then centrifuged at 3000×gfor 10 minutes. After centrifuge, the top layer was transferred into a15-mL tube using positive displacement pipettes. The oil was extractedfrom the slurry again with the same method. The total weight oftransferred liquor was measured. The oil content and density weremeasured on densiometer. The final extracted oil was calculated as:

Extracted oil (g/flask)=first extracted oil (g/flask)+second extractedoil (g/flask)

Compared with the control strain MHCT-484, both lipase-expressing yeaststrains HP21-F04 and HP21-F05 improved the final oil yield (0.518g/flask and 0.966 g/flask for strains HP21-F04 and HP21-F05,respectively compared to 0.483 g/flask for control strain MHCT-484).

Example 9: Defoaming Capabilities with Phospholipase-Expressing YeastStrains

Commercial Amp corn mash was obtained from Trenton Agri at 35.9% (w/w)of dry solids content and was diluted with tape water to 32.0% (w/w).After dilution, the pH value of the mash was adjusted to 5.1 with 39%(w/v) NaOH solution. Urea and lactrol were added into the pH adjustedAmp mash to final concentration at 150 ppm and 3 ppm, respectively. Theprepared corn mash was aliquoted into 250-mL flasks (100 g/flask).

To propagate yeast, 50 mL of 6% YPD and 100 μL of yeast-glycerol stocksolution were mixed in a 125-mL flask and then incubated at 32° C. forovernight. After incubation, 45 mL of propagation was transferred to a50-mL centrifuge tube and centrifuged at 3500 rpm for 10 minutes. Theliquid fraction was decanted, and deionized water was used to twice washthe cells. The cells were resuspended in 10 mL of deionized water andthe total and dead cell accounts were measured using a NucleoCounter®YC-100.

The exogenous α-glucoamylase, Spirizyme Achieve-T™ (Novozyems A/S), wasadded into the flasks containing corn mash per DOE and mixed well. Thenthe pre-determined amount of yeast suspension was added and mixed well.Also, the pre-determined exogenous lipase was added and used as control.The fermentation was performed at 32° C. for 54 hours.

The defoaming capability of control MHCT-484 and lipase-expressing yeastHP21-F04 was monitored using video camera after 12-hour fermentation.Compared with control yMHCT48, lipase-expressing yeast HP21-F04 showedsignificant defoaming capability (FIG. 4).

1-20. (canceled) 21: A method of producing a fermentation product from astarch-containing or cellulosic-containing material comprising: (a)saccharifying the starch-containing or cellulosic-containing material;and (b) fermenting the saccharified material of step (a) with afermenting organism; wherein the fermenting organism comprises aheterologous polynucleotide encoding a phospholipase. 22: The method ofclaim 21, wherein the phospholipase is a Phospholipase A or aPhospholipase C. 23: The method of claim 21, wherein the phospholipasehas a mature polypeptide sequence with at least 80% sequence identity tothe amino acid sequence of any one of SEQ ID NOs: 235-242 and 252-342.24: The method of claim 21, wherein saccharification of step (a) occurson a starch-containing material, and wherein the starch-containingmaterial is either gelatinized or ungelatinized starch. 25: The methodof claim 24, comprising liquefying the starch-containing material bycontacting the material with an alpha-amylase prior to saccharification.26: The method of claim 25, wherein liquefying the starch-containingmaterial and/or saccharifying the starch-containing material isconducted in presence of exogenously added protease. 27: The method ofclaim 21, wherein fermentation is performed under reduced nitrogenconditions. 28: The method of claim 21, wherein the fermenting organismcomprises a heterologous polynucleotide encoding a glucoamylase. 29: Themethod of claim 21, wherein the fermenting organism comprises aheterologous polynucleotide encoding an alpha-amylase. 30: The method ofclaim 21, wherein the fermenting organism comprises a heterologouspolynucleotide encoding a protease. 31: The method of claim 21, whereinthe fermenting organism is a Saccharomyces cerevisiae cell. 32: Themethod of claim 21, wherein the method results in higher yield offermentation product and/or reduced foam accumulation when compared tothe same process using an identical cell without the heterologouspolynucleotide encoding the phospholipase under the same conditions. 33:The method of claim 1, wherein the method results in at least 0.25%higher yield of fermentation product. 34: A recombinant yeast cellcomprising a heterologous polynucleotide encoding a phospholipase. 35:The recombinant yeast cell of claim 34, wherein the phospholipase is aPhospholipase A or a Phospholipase C. 36: The recombinant yeast cell ofclaim 34, wherein the phospholipase has a mature polypeptide sequence atleast 80% sequence identity, to the amino acid sequence of any one ofSEQ ID NOs: 235-242 and 252-342. 37: The recombinant yeast cell of claim34, wherein the fermenting organism comprises a heterologouspolynucleotide encoding a glucoamylase. 38: The recombinant yeast cellof claim 34, wherein the fermenting organism comprises a heterologouspolynucleotide encoding an alpha-amylase. 39: The recombinant yeast cellof claim 34, wherein the fermenting organism comprises a heterologouspolynucleotide encoding a protease. 40: The recombinant yeast of claim34, wherein the cell is a Saccharomyces cerevisiae cell.